Inhibition of platelet aggregation using anti-human GPVI antibodies

ABSTRACT

The present invention relates to an isolated humanized protein binding to human Glycoprotein VI (hGPVI) for treating a GPVI-related condition in a subject in need thereof, wherein said isolated humanized protein is to be administered during at least 2 hours to the subject, preferably during at least 4 to 6 hours.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 filing of International PatentApplication No. PCT/EP2018/052664, filed Feb. 2, 2018, which claimspriority to European Patent Application No. 17154658.3, filed Feb. 3,2017, the entire disclosures of which are hereby incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to the treatment of cardiovasculardiseases. In particular, the present invention relates to a method fortreating cardiovascular disease comprising the continuous administrationof a novel anti-human glycoprotein VI antibody or fragment thereof to ahuman patient in need thereof.

BACKGROUND OF INVENTION

Acute coronary and cerebrovascular accidents are currently a major causeof death in the world. In addition, the global incidence of recurrenceand death in the 6-month post-treatment period after an acute coronarysyndrome is still 8-15%.

In the case of acute coronary syndrome with ST segment elevation,mechanical treatment with coronary angioplasty and introduction of astent is highly efficient to urgently restore coronary artery flow, butdoes not prevent morbidity/mortality for about 15% of patients in thenext 6 months. Thrombolytic treatments, which are based on long termfibrinolytic, anticoagulant and anti-aggregating drugs associations,give even less encouraging results. Indeed, despite improvements inmedical treatment of thrombosis, morbidity/mortality at 6 months issimilar to that observed for acute coronary syndrome without ST segmentelevation.

Regarding cerebrovascular ischemic accidents, treatments are still verylimited due to the generally late caring of most patients and to thehemorrhagic risk of currently available anti-thrombotic treatments.

There is thus still a clinical need for improving treatments forcardiovascular diseases, and especially for new molecules with improvedfeatures compared to available molecules and for specific dosageregimens for administering said molecules. The challenge to face is toobtain molecules and protocols of administration with excellentefficiency on the pathological thrombosis but devoid of risk ofbleeding.

To answer to this requirement, the target must have a greater role inthrombus formation occurring in a diseased vessel than in physiologicalhemostasis required to limit the bleeding in a healthy vessel. This isthe case of platelet Glycoprotein VI that has been demonstrated inanimals to play a role in experimental thrombosis including stroke,vascular remodeling and to be critical in atherothrombosis.

Contrary to αIIbβ3 integrin antagonists, which are currently used inthrombosis treatment and inhibit platelet final activation phase, i.e.,platelet aggregation, and to antagonists of platelet recruitment(inhibitors of the P2Y12 ADP receptor and aspirin), GPVI is implicatedinto several steps of the platelet plug formation: initiation via theinteraction with the injured vascular wall, amplification via initialplatelet activation leading to the secretion of secondary agonists, theactivation of integrins and of platelet procoagulant activity, growthand stabilization via the interaction with fibrin (Mammadova-Bach etal., 2015. Blood. 126(5):683-91). Thus, GPVI antagonists may prevent notonly platelet aggregation, but also secondary agonists liberation aswell as growth factors and cytokines secretion resulting into vascularlesions development. Finally, GPVI expression is limited to plateletsand megakaryocytes, and thus represents a perfectly specific target foranti-thrombosis treatment.

GPVI antagonists were thus developed for treating cardiovasculardiseases.

WO 2001/000810 and WO 2003/008454 both describe a soluble GPVIrecombinant protein which is a fusion protein between the GPVIextracellular domain and a human Ig Fc domain. This soluble recombinantGPVI protein competes with platelet GPVI for binding collagen.Encouraging results were first obtained with this soluble GPVI proteinin a thrombosis murine model, but these results were not confirmed. Inaddition, this approach involves structural, functional andpharmacological disadvantages. First, this compound is a high molecularweight chimeric protein (˜160 kDa). GPVI-Fc targets the collagen exposedat the site of the vascular injury, the amount and accessibility ofwhich are poorly predictable and, thus, the amount of product to beinjected constitutes a potential limitation to the use of GPVI-Fc.Another limitation could be the risk of immunization against neoepitopespotentially exposed on the fusion protein.

Neutralizing monoclonal antibodies directed against human GPVI were alsodescribed in the art.

For example, EP 1224942 and EP 1228768 disclose the rat monoclonalanti-GPVI antibody JAQ1, which specifically binds to mouse GPVI, for thetreatment of thrombotic disease. JAQ1 antibody induces irreversibleinternalization of the GPVI receptor on mouse platelets.

EP 1538165 describes another rat monoclonal anti-GPVI antibody (hGP5C4), which Fab fragment was found to have marked inhibitory effects onthe main physiological functions of platelets induced by collagenstimulation: stimulation of collagen-mediated physiological activationmarkers PAC-I and CD62P-Selectin was completely prevented by hGP 5C4Fab, and hGP 5C4 Fab potently inhibited human platelet aggregation exvivo without any intrinsic activity. However, 5C4 is a rat antibody, andtherefore only presents a very limited therapeutic potential.

WO 2005/111083 describes 4 mouse monoclonal anti-GPVI antibodies OM1,OM2, OM3 and OM4, that were found to inhibit GPVI binding to collagen,collagen-induced secretion and thromboxane A2 (TXA2) formation in vitro,as well as ex vivo collagen-induced platelet aggregation afterintravenous injection to Cynomolgus monkeys. OM4 also appears to inhibitthrombus formation in a rat thrombosis model.

WO 2001/000810 also describes various murine monoclonal anti-GPVIantibodies named 8M14.3, 3F8.1, 9E18.3, 3J24.2, 6E12.3, 1P10.2, 4L7.3,7H4.6, 9012.2, 7H14.1, and 9E18.2, as well as several human phageantibodies scFv fragments named A9, A10, C9, A4, C10, B4, C3 and D11.Some of these antibodies and scFv fragments were found to inhibit GPVIbinding to collagen, including antibodies 9E18.3, 7H4.6, and 9O12.2, andscFv fragments A10, A4, C10, B4, C3 and D11. In addition, 9O12.2 Fabfragments were found to completely block collagen-induced plateletaggregation and secretion, to block fibrin-induced platelet aggregation,to inhibit the procoagulant activity of collagen-stimulated orfibrin-stimulated platelets and platelet adhesion to collagen or fibrinin static conditions, to impair platelet adhesion and to prevent thrombiformation under arterial flow conditions.

WO 2008/049928 describes a scFv fragment derived from 9O12.2,constituted of the VH and VL domains of 9O12.2 monoclonal antibodylinked via a (Gly₄Ser)₃ peptide.

However, none of the currently known anti-GPVI antibodies was shown tobe efficient in vivo for preventing and/or treating cardiovasculardiseases. In particular, the majority of anti-GPVI antibodies that havebeen reported appeared not fitted for the development of anantithrombotic for medical use in human, especially due to their animalorigin.

In particular, some human phage antibodies scFvs directed to human GPVIhave been reported to be inhibitory but their affinity appears to below. Moreover, cross-linking of GPVI at the platelet surface by adivalent or multivalent ligand such as 9O12.2 whole IgG results inplatelet activation via GPVI dimerization and via cross-linking of GPVIto the low affinity Fc receptor (FcγRIIA). In contrast, monovalent9O12.2 Fab and scFv fragments are inhibitory.

However, these fragments could not be used in therapeutic due to theirsize and to their animal origin which makes them immunogenic in humanpatients. Moreover, scFv fragments present a short half-life, whichlimits their therapeutic potentials.

There is thus still a need for neutralizing GPVI antagonists withoutimmunogenicity in human.

US 2006/0088531 describes a human scFv fragment 10B12, presenting aK_(D) for binding to human GPVI of about 7.9·10⁻⁷ M. Smethurst(Smethurst et al., 2004. Blood. 103(3):903-11) further describes theepitope bound by 10B12 on the Ig-like C2-type domain 1 (D1) of humanGPVI. This epitope comprises residues R58, K61, R66, K79 and R80 ofhuman GPVI (numbering based on UniProtKB accession number Q9HCN6).

O'Connor (O'Connor et al., 2006. J. Biol. Chem. 281(44):33505-10) alsodescribes a human scFv fragment 1C3, of low affinity for GPVI (5.4 10⁻⁷M), which neither blocked collagen-induced platelet aggregation nor GPVIbinding to collagen but which potentiated the inhibitory effect of the10B12 antibody. The 1C3 epitope in GPVI comprises amino acid 1168, andit is further postulated that this epitope might encompass a regionbetween residues S164 and S182, a region which is highly conserved frommouse to human.

There is thus a need for a humanized antibody (i.e., an antibody withoutimmunogenicity in human) with improved affinity, efficacy and half-lifeas compared to the antagonists of the prior art, as a means forefficiently and contentedly preventing and/or treating cardiovasculardiseases in human. Moreover, these antagonists should preferably beeasily purified.

In the art, anti-GPVI antibodies inducing a GPVI depletion phenotypewere described (WO 2006/118350, WO 2011/073954, EP 2363416). Inparticular, WO 2006/118350 discloses an anti-GPVI antibody directedagainst all mammalians. The epitope of GPVI bound by this antibody isdescribed and corresponds to the loops 9 and 11 of the Ig-like C2-typedomain 2 of GPVI, corresponding to amino acid residues 136-142 and158-162, respectively.

However, GPVI depletion is undesirable since it cannot be controlled,and is irreversible (i.e., it lasts the lifetime of platelets, or evenlonger due to GPVI depletion on megakaryocytes).

In therapy, a rapid, safe and prolonged (in the range of hours)antiplatelet effect is required. Therefore, antibodies not inducing aGPVI depletion phenotype, and preferably not inducing a decrease inplatelet count in vivo (i.e., with a reversible effect) should bedeveloped.

The Applicant developed an antibody anti-GPVI, wherein said antibody orthe antigen binding fragment thereof binds to a novel, undescribed,conformational epitope. Said antibody anti-GPVI has a strong affinityfor human GPVI, and blocks GPVI interaction with its ligands (includingcollagen and fibrin), without decreasing the platelet count nordepleting GPVI in vivo. When continuously administered for at least 2hours, the antibody (or a fragment thereof) demonstrates a prolonged invivo effect. The present invention thus relates to the therapeutic useof an improved humanized neutralizing antibody specific to human GPVI ora fragment thereof.

SUMMARY

The present invention relates to an isolated humanized protein bindingto human Glycoprotein VI (hGPVI) for treating a GPVI-related conditionin a subject in need thereof, wherein said isolated humanized protein isto be administered during at least 2 hours to the subject, preferablyduring at least 4 to 6 hours.

In one embodiment, the isolated humanized protein is injected,preferably by intravenous infusion. In another embodiment, the isolatedhumanized protein is injected intraperitoneally.

In one embodiment, a dose of humanized protein ranging from about 0.5mg/kg to about 50 mg/kg, preferably from about 1 mg/kg to about 32mg/kg, more preferably from about 2.5 mg/kg to about 25 mg/kg, even morepreferably from about 5 mg/kg to about 15 mg/kg, and still even morepreferably of about 8 mg/kg is to be administered to the patient.

In another embodiment, a dose of the protein for use in the presentinvention ranges from about 125 mg to about 2000 mg, preferably fromabout 250 mg to about 1000 mg or from about 500 mg to about 1000 mg.

In one embodiment, a first bolus is administered, wherein preferablysaid first bolus administration comprises about 10 to 50%, preferablyabout 20% of the total dosage of the isolated humanized protein to beadministered, and wherein preferably said first bolus is administered inabout 5 to 30 minutes, preferably in about 15 minutes.

In one embodiment, said protein binds to a conformational epitopecomprising:

-   -   at least one amino acid residue from amino acid residues 114 to        142 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least        60% of identity over amino acid residues 114 to 142 of hGPVI        (SEQ ID NO: 13); and    -   at least one amino acid residue from amino acid residues 165 to        187 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least        60% of identity over amino acid residues 165 to 187 of hGPVI        (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least oneamino acid residue from amino acid residues 121 to 135 of hGPVI (SEQ IDNO: 13) or from a sequence sharing at least 60% of identity over aminoacid residues 121 to 135 of hGPVI (SEQ ID NO: 13); and at least oneamino acid residue from amino acid residues 169 to 183 of hGPVI (SEQ IDNO: 13) or from a sequence sharing at least 60% of identity over aminoacid residues 169 to 183 of hGPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least oneamino acid residue from amino acid residues 121 to 136 of hGPVI (SEQ IDNO: 13) or from a sequence sharing at least 60% of identity over aminoacid residues 121 to 136 of hGPVI (SEQ ID NO: 13); and at least oneamino acid residue from amino acid residues 169 to 183 of hGPVI (SEQ IDNO: 13) or from a sequence sharing at least 60% of identity over aminoacid residues 169 to 183 of hGPVI (SEQ ID NO: 13).

In one embodiment, said protein has a K_(D) for binding to hGPVI lessthan 15 nM, wherein said K_(D) is measured by surface plasmon resonanceusing 960 to 1071 RU of soluble human GPVI and using PBS pH 7.4 asrunning buffer and wherein said isolated humanized protein does notinduce a GPVI depletion phenotype in vivo.

In one embodiment, the isolated humanized protein binding to hGPVI foruse according to the present invention is an antibody molecule selectedfrom the group consisting of a whole antibody, a humanized antibody, asingle chain antibody, a Fv, a Fab; or an antibody fragment selectedfrom the group consisting of a unibody, a domain antibody, and ananobody; or a monomeric antibody mimetic selected from the groupconsisting of an affibody, an affilin, an affitin, an adnectin, anatrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, and aversabody, preferably a monovalent antibody.

In one embodiment, the isolated antibody molecule binding to hGPVI is anantibody molecule wherein:

-   -   the variable region of the heavy chain comprises at least one of        the following CDRs:

VH-CDR1: (SEQ ID NO: 1) GYTFTSYNM; VH-CDR2: (SEQ ID NO: 2)GIYPGNGDTSYNQKFQG; and VH-CDR3: (SEQ ID NO: 3) GTVVGDWYFDV;

-   -   or any CDR having an amino acid sequence that shares at least        60% of identity with SEQ ID NO: 1-3; and    -   the variable region of the light chain comprises at least one of        the following CDRs:

VL-CDR1: (SEQ ID NO: 4) RSSQSLENSNGNTYLN; VL-CDR2: (SEQ ID NO: 5)RVSNRFS; and VL-CDR3: (SEQ ID NO: 6) LQLTHVPWT;

-   -   or any CDR having an amino acid sequence that shares at least        60% of identity with SEQ ID NO: 4-6.

In one embodiment, the variable region of the heavy chain comprises thefollowing CDRs: GYTFTSYNMH (SEQ ID NO: 1), GIYPGNGDTSYNQKFQG (SEQ ID NO:2) and GTVVGDWYFDV (SEQ ID NO: 3) and the variable region of the lightchain comprises the following CDRs: RSSQSLENSNGNTYLN (SEQ ID NO: 4),RVSNRFS (SEQ ID NO: 5) and LQLTHVPWT (SEQ ID NO: 6) or any CDR having anamino acid sequence that shares at least 60% of identity with said SEQID NO: 1-6.

In one embodiment, the amino acid sequence encoding the heavy chainvariable region is SEQ ID NO: 7 and the amino acid sequence encoding thelight variable region is SEQ ID NO: 8, or any sequence having an aminoacid sequence that shares at least 60% of identity with said SEQ ID NO:7 or 8.

In one embodiment, the amino acid sequence encoding the heavy chainvariable region is SEQ ID NO: 7 and the amino acid sequence encoding thelight variable region is SEQ ID NO: 9, or any sequence having an aminoacid sequence that shares at least 60% of identity with said SEQ ID NO:7 or 9.

In one embodiment, said GPVI-related condition is a cardiovasculardisease selected from arterial and venous thrombosis, restenosis, acutecoronary syndrome and cerebrovascular accidents due to atherosclerosis.

In one embodiment, said GPVI-related condition is a cardiovasculardisease selected from arterial and venous thrombosis, restenosis, acutecoronary syndrome, cerebrovascular accidents due to atherosclerosis,myocardial infarction, pulmonary embolism, critical limb ischemia andperipheral artery disease.

DETAILED DESCRIPTION

“Glycoprotein VI (GPVI)” is a platelet membrane glycoprotein that isinvolved in platelet-collagen interactions. GPVI is a transmembranecollagen receptor expressed on the surface of platelets. In oneembodiment, the amino acid sequence of human GPVI is SEQ ID NO: 13(accession number: BAA89353.1) or any amino acid sequence presenting atleast about 90% identity with SEQ ID NO: 13, preferably at least about91, 92, 93, 94, 95, 96, 97, 98, 99% identity or more with SEQ ID NO: 13.

(SEQ ID NO: 13) MSPSPTALFCLGLCLGRVPA (Signal peptide).QSGPLPKPSLQALPSSLVPLEKPVTLRCQGPPGVDLYRLEKLSSSRYQDQAVLFIPAMKRSLAGRYRCSYQNGSLWSLPSDQLELVATGVFAKPSLSAQPGPAVSSGGDVTLQCQTRYGFDQFALYKEGDPAPYKNPERWYRASFPIITVTAAHSGTYRCYSFSSRDPYLWSAPSDPLELVVTGTSVTPSRLPTEPPSSVAEFSEATAELTVSFTNKVFTTETSRSITTSPKESDSPAGPARQYYTKGN  (Extracellular domain).LVRICLGAVILIILAGFLAEDWHSRRKRLRHRGRAVQRPLPPLPPLPQTRKSHGGQDGGRQDVHSRGLCS (Transmembrane and cytoplasmic domains).

The extracellular domain of GPVI is composed of two Ig-like C2-typedomains, namely D1 and D2, linked by a hinge interdomain. In oneembodiment, D1 comprises amino acid residues 21 to 109 of SEQ ID NO: 13.In one embodiment, the hinge interdomain between D1 and D2 comprisesamino acid residues 110 to 113 of SEQ ID NO: 13. In one embodiment, D2comprises amino acid residues 114 to 207 of SEQ ID NO: 13.

“About” preceding a figure means plus or less 10% of the value of saidfigure.

“Antibody” or “Immunoglobulin”—As used herein, the term “immunoglobulin”includes a protein having a combination of two heavy and two lightchains whether or not it possesses any relevant specificimmunoreactivity. “Antibodies” refers to such assemblies which havesignificant known specific immunoreactive activity to an antigen ofinterest (e.g. human GPVI). The term “anti-GPVI antibodies” is usedherein to refer to antibodies which exhibit immunological specificityfor human GPVI protein. As explained elsewhere herein, “specificity” forhuman GPVI does not exclude cross-reaction with species homologues ofGPVI. Antibodies and immunoglobulins comprise light and heavy chains,with or without an interchain covalent linkage between them. Basicimmunoglobulin structures in vertebrate systems are relatively wellunderstood. The generic term “immunoglobulin” comprises five distinctclasses of antibody that can be distinguished biochemically. All fiveclasses of antibodies are within the scope of the present invention; thefollowing discussion will generally be directed to the IgG class ofimmunoglobulin molecules. With regard to IgG, immunoglobulins comprisetwo identical light polypeptide chains of molecular weight of about23,000 Daltons, and two identical heavy chains of molecular weight ofabout 53,000-70,000 Daltons. The four chains are joined by disulfidebonds in a “Y” configuration wherein the light chains bracket the heavychains starting at the mouth of the “Y” and continuing through thevariable region. The light chains of an antibody are classified aseither kappa or lambda ([K], [λ]). Each heavy chain class may be bondedwith either a kappa or lambda light chain. In general, the light andheavy chains are covalently bonded to each other, and the “tail” regionsof the two heavy chains are bonded to each other by covalent disulfidelinkages or non-covalent linkages when the immunoglobulins are generatedeither by hybridomas, B cells or genetically engineered host cells. Inthe heavy chain, the amino acid sequences run from an N-terminus at theforked ends of the Y configuration to the C-terminus at the bottom ofeach chain. Those skilled in the art will appreciate that heavy chainsare classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε)with some subclasses among them (e.g., γ1-γ4). It is the nature of thischain that determines the “class” of the antibody as IgG, IgM, IgA IgD,or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g.,IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are knownto confer functional specialization. Modified versions of each of theseclasses and isotypes are readily discernable to the skilled artisan inview of the instant disclosure and, accordingly, are within the scope ofthe instant invention. As indicated above, the variable region of anantibody allows the antibody to selectively recognize and specificallybind epitopes on antigens. That is, the light chain variable domain (VLdomain) and heavy chain variable domain (VH domain) of an antibodycombine to form the variable region that defines a three-dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site presents at the end of each arm of the “Y”. Morespecifically, the antigen binding site is defined by threecomplementarity determining regions (CDRs) on each of the VH and VLchains.

“An isolated antibody”—As used herein, an “isolated antibody” is onethat has been separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials that would interfere with diagnostic or therapeutic uses ofthe antibody, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous components. In preferred embodiments, the antibodyis purified: (1) to greater than 80, 85, 90, 91, 92, 93, 94, 95% or moreby weight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight; (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator; or (3) to homogeneity as shown bySDS-PAGE under reducing or non-reducing conditions and using Coomassieblue or, preferably, silver staining. Isolated antibody includes theantibody in situ within recombinant cells since at least one componentof the antibody's natural environment will not be present. Ordinarily,however, isolated antibody will be prepared by at least one purificationstep.

“Affinity variants”—As used herein, the term “affinity variant” refersto a variant antibody which exhibits one or more changes in amino acidsequence compared to a reference anti-GPVI antibody, wherein theaffinity variant exhibits an altered affinity for the human GPVI proteinin comparison to the reference antibody. Typically, affinity variantswill exhibit an improved affinity for human GPVI, as compared to thereference anti-GPVI antibody. The improvement may be either a lowerK_(D) for human GPVI, a higher K_(A) for human GPVI, a faster on-ratefor human GPVI or a slower off-rate for human GPVI or an alteration inthe pattern of cross-reactivity with non-human GPVI homologues. Affinityvariants typically exhibit one or more changes in amino acid sequence(such as, for example in the CDRs), as compared to the referenceanti-GPVI antibody. Such substitutions may result in replacement of theoriginal amino acid present at a given position in the CDRs with adifferent amino acid residue, which may be a naturally occurring aminoacid residue or a non-naturally occurring amino acid residue. The aminoacid substitutions may be conservative or non-conservative.

“Binding Site”—As used herein, the term “binding site” comprises aregion of a protein which is responsible for selectively binding to atarget antigen of interest (e.g. human GPVI). Binding domains or bindingregions comprise at least one binding site. Exemplary binding domainsinclude an antibody variable domain. The protein of the invention maycomprise a single antigen binding site or multiple (e.g., two, three orfour) antigen binding sites. Preferably, however, the protein of theinvention comprises a single antigen binding site.

“Conservative amino acid substitution”—As used herein, “conservativeamino acid substitutions” are ones in which the amino acid residue isreplaced with an amino acid residue that has similar properties, suchthat one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Amino acid substitutions may further be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Other families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in an immunoglobulinpolypeptide may be replaced with another amino acid residue from thesame side chain family. In another embodiment, a string of amino acidscan be replaced with a structurally similar string that differs in orderand/or composition of side chain family members.

“Chimeric”—As used herein, a “chimeric” protein comprises a first aminoacid sequence linked to a second amino acid sequence with which it isnot naturally linked in nature. The amino acid sequences may normallyexist in separate proteins that are brought together in the fusionprotein or they may normally exist in the same protein but are placed ina new arrangement in the fusion protein. A chimeric protein may becreated, for example, by chemical synthesis, or by creating andtranslating a polynucleotide in which the peptide regions are encoded inthe desired relationship.

“CDR”—As used herein, the term “CDR” or “complementarity determiningregion” means the non-contiguous antigen combining sites found withinthe variable region of both heavy and light chain polypeptides. CDRswere identified according to the following rules as deduced from Kabat(Kabat et al., 1991. J. Immunol. 147(5):1709-19) and Chothia & Lesk(Chothia C. and A. M. Lesk, 1987. J. Mol. Biol. 196(4):901-17):

-   -   CDR-L1:    -   Start—Approx residue 24;    -   Residue before is always a Cys;    -   Residue after is always a Trp. Typically TRP-TYR-GLN, but also,        TRP-LEU-GLN, TRP-PHE-GLN, TRP-TYR-LEU;    -   Length 10 to 17 residues;    -   CDR-L2:    -   Start—always 16 residues after the end of L1;    -   Residues before generally ILE-TYR, but also, VAL-TYR, ILE-LYS,        ILE-PHE;    -   Length always 7 residues;    -   CDR-L3:    -   Start—always 33 residues after end of L2;    -   Residue before is always Cys;    -   Residues after always PHE-GLY-XXX-GLY (SEQ ID NO: 21);    -   Length 7 to 11 residues;    -   CDR-H1:    -   Start—Approx residue 26 (always 4 after a CYS) [Chothia/AbM        definition] Kabat definition starts 5 residues later;    -   Residues before always CYS-XXX-XXX-XXX (SEQ ID NO: 22);    -   Residues after always a TRP. Typically TRP-VAL, but also,        TRP-ILE, TRP-ALA    -   Length 10 to 12 residues (AbM definition) Chothia definition        excludes the last 4 residues;    -   CDR-H2:    -   Start—always 15 residues after the end of Kabat/AbM definition)        of CDR-H1 Residues before typically LEU-GLU-TRP-ILE-GLY (SEQ ID        NO: 23), but a number of variations;    -   Residues after LYS/ARG-LEU/ILE/VAL/PHE/THR/ALA-THR/SER/ILE/ALA        Length Kabat definition 16 to 19 residues (AbM definition ends 7        residues earlier);    -   CDR-H3:    -   Start—always 33 residues after end of CDR-H2 (always 2 after a        CYS);    -   Residues before always CYS-XXX-XXX (typically CYS-ALA-ARG);    -   Residues after always TRP-GLY-XXX-GLY (SEQ ID NO: 24);    -   Length 3 to 25 residues.

“CH2 domain”—As used herein, the term “CH2 domain” includes the regionof a heavy chain molecule that usually extends from about amino acid 231to about amino acid 340. The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It has been speculated that the carbohydratemay provide a substitute for the domain-domain pairing and helpstabilize the CH2 domain (Burton, Molec. Immunol. 22 (1985) 161-206).

“Derived from”—As used herein, the term “derived from” a designatedprotein (e.g., an anti-GPVI antibody or antigen-binding fragmentthereof) refers to the origin of the protein. In an embodiment, theprotein or amino acid sequence which is derived from a particularstarting protein is a CDR sequence or sequence related thereto. In anembodiment, the amino acid sequence which is derived from a particularstarting protein is not contiguous. For example, in an embodiment, one,two, three, four, five, or six CDRs are derived from a startingantibody. In an embodiment, the protein or amino acid sequence which isderived from a particular starting protein or amino acid sequence has anamino acid sequence that is essentially identical to that of thestarting sequence, or a region thereof wherein the region consists of atleast 3-5 amino acids, at least 5-10 amino acids, at least 10-20 aminoacids, at least 20-30 amino acids, or at least 30-50 amino acids, orwhich is otherwise identifiable to one of ordinary skill in the art ashaving its origin in the starting sequence. In an embodiment, the one ormore CDR sequences derived from the starting antibody are altered toproduce variant CDR sequences, e.g., affinity variants, wherein thevariant CDR sequences maintain GPVI binding activity.

“Diabodies”—As used herein, the term “diabodies” refers to smallantibody fragments prepared by constructing sFv fragments (see sFvparagraph) with short linkers (about 5-residues) between the VH and VLdomains such that inter-chain but not intra-chain pairing of the Vdomains is achieved, resulting in a bivalent fragment, i.e., fragmenthaving two antigen-binding sites. Bispecific diabodies are heterodimersof two “crossover” sFv fragments in which the VH and VL domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 0404097; WO 1993/011161;and Holliger (Holliger et al., 1993. Proc. Natl. Acad. Sci.90(14):6444-6448).

“Engineered”—As used herein, the term “engineered” includes manipulationof nucleic acid or polypeptide molecules by synthetic means (e.g., byrecombinant techniques, in vitro peptide synthesis, by enzymatic orchemical coupling of peptides or some combination of these techniques).Preferably, the antibodies of the invention are engineered, includingfor example, humanized and/or chimeric antibodies, and antibodies whichhave been engineered to improve one or more properties, such as antigenbinding, stability/half-life or effector function.

“Epitope”—As used herein, the term “epitope” refers to a specificarrangement of amino acids located on a protein or proteins to which anantibody binds. Epitopes often consist of a chemically active surfacegrouping of molecules such as amino acids or sugar side chains, and havespecific three dimensional structural characteristics as well asspecific charge characteristics. Epitopes can be linear orconformational, i.e., involving two or more sequences of amino acids invarious regions of the antigen that may not necessarily be contiguous.

“Framework region”—As used herein, the term “framework region” or “FRregion” includes the amino acid residues that are part of the variableregion, but are not part of the CDRs (e.g., using the Kabat/Chothiadefinition of CDRs). Therefore, a variable region framework is betweenabout 100-120 amino acids in length but includes only those amino acidsoutside of the CDRs. For the specific example of a heavy chain variableregion and for the CDRs as defined by Kabat/Chothia, framework region 1may correspond to the domain of the variable region encompassing aminoacids 1-25; framework region 2 may correspond to the domain of thevariable region encompassing amino acids 36-49; framework region 3 maycorrespond to the domain of the variable region encompassing amino acids67-98, and framework region 4 may correspond to the domain of thevariable region from amino acids 110 to the end of the variable region.The framework regions for the light chain are similarly separated byeach of the light chain variable region CDRs. In naturally occurringantibodies, the six CDRs present on each monomeric antibody are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding site as the antibody assumes its threedimensional configuration in an aqueous environment. The remainders ofthe heavy and light variable domains show less inter-molecularvariability in amino acid sequence and are termed the framework regions.The framework regions largely adopt a [beta]-sheet conformation and theCDRs form loops which connect, and in some cases form part of, the[beta]-sheet structure. Thus, these framework regions act to form ascaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding site formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope. The position of CDRs can bereadily identified by one of ordinary skill in the art.

“Fragment”—As used herein, the term “fragment” refers to a part orregion of an antibody or antibody chain comprising fewer amino acidresidues than an intact or complete antibody or antibody chain. The term“antigen-binding fragment” refers to a protein fragment of animmunoglobulin or antibody that binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding to human GPVI). As usedherein, the term “fragment” of an antibody molecule includesantigen-binding fragments of antibodies, for example, an antibody lightchain variable domain (VL), an antibody heavy chain variable domain(VH), a single chain antibody (scFv), a F(ab′)₂ fragment, a Fabfragment, an Fd fragment, an Fv fragment, a single domain antibodyfragment (DAb), a one-armed (monovalent) antibody, diabodies or anyantigen-binding molecule formed by combination, assembly or conjugationof such antigen binding fragments. Fragments can be obtained, e.g., viachemical or enzymatic treatment of an intact or complete antibody orantibody chain or by recombinant means.

The “Fc” fragment of an antibody comprises the carboxy-terminal portionsof both H chains held together by disulfides. The effector functions ofantibodies are determined by sequences in the Fc region, which region isalso the part recognized by Fc receptors (FcR) found on certain types ofcells.

“Fv”—As used herein, the term “Fv” is the minimum antibody fragment thatcontains a complete antigen-recognition and-binding site. This fragmentconsists of a dimer of one heavy- and one light-chain variable regiondomain in tight, non-covalent association. From the folding of these twodomains emanate six hypervariable loops (three loops each from the H andL chain) that contribute to antigen binding and confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

“Heavy chain region”—As used herein, the term “heavy chain region”includes amino acid sequences derived from the constant domains of animmunoglobulin heavy chain. A protein comprising a heavy chain regioncomprises at least one of: a CH1 domain, a hinge (e.g., upper, middle,and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or avariant or fragment thereof. In an embodiment, a binding molecule of theinvention may comprise the Fc region of an immunoglobulin heavy chain(e.g., a hinge portion, a CH2 domain, and a CH3 domain). In anotherembodiment, a binding molecule of the invention lacks at least a regionof a constant domain (e.g., all or part of a CH2 domain). In certainembodiments, at least one, and preferably all, of the constant domainsare derived from a human immunoglobulin heavy chain. For example, in onepreferred embodiment, the heavy chain region comprises a fully humanhinge domain.

In other preferred embodiments, the heavy chain region comprising afully human Fc region (e.g., hinge, CH2 and CH3 domain sequences from ahuman immunoglobulin). In certain embodiments, the constituent constantdomains of the heavy chain region are from different immunoglobulinmolecules. For example, a heavy chain region of a protein may comprise aCH2 domain derived from an IgG1 molecule and a hinge region derived froman IgG3 or IgG4 molecule. In other embodiments, the constant domains arechimeric domains comprising regions of different immunoglobulinmolecules. For example, a hinge may comprise a first region from an IgG1molecule and a second region from an IgG3 or IgG4 molecule. As set forthabove, it will be understood by one of ordinary skill in the art thatthe constant domains of the heavy chain region may be modified such thatthey vary in amino acid sequence from the naturally occurring(wild-type) immunoglobulin molecule. That is, the proteins of theinvention disclosed herein may comprise alterations or modifications toone or more of the heavy chain constant domains (CH1, hinge, CH2 or CH3)and/or to the light chain constant domain (CL). Exemplary modificationsinclude additions, deletions or substitutions of one or more amino acidsin one or more domains.

“Hinge region”—As used herein, the term “hinge region” includes theregion of a heavy chain molecule that joins the CH1 domain to the CH2domain. This hinge region comprises approximately 25 residues and isflexible, thus allowing the two N-terminal antigen binding regions tomove independently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., 1998. JImmunol. 161(8):4083-90).

The terms “hypervariable loop” and “complementarity determining region”are not strictly synonymous, since the hypervariable loops (HVs) aredefined on the basis of structure, whereas complementarity determiningregions (CDRs) are defined based on sequence variability (Kabat, ElvinA. (1983). Sequences of proteins of immunological interest (5thedition). Besthesda, Md.: Public Health Service, National Institutes ofHealth) and the limits of the HVs and the CDRs may be different in someVH and VL domains. The CDRs of the VL and VH domains can typically bedefined by the Kabat/Chothia definition (see above). In one embodiment,the CDRs of the VL and VH domains may comprise the following aminoacids: residues 24-39 (CDRL1), 55-61 (CDRL2) and 94-102 (CDRL3) in thelight chain variable domain, and residues 26-35 (CDRH1), 50-66 (CDRH2)and 99-109 (CDRH3) in the heavy chain variable domain. Thus, the HVs maybe comprised within the corresponding CDRs and references herein to the“hypervariable loops” of VH and VL domains should be interpreted as alsoencompassing the corresponding CDRs, and vice versa, unless otherwiseindicated. The more highly conserved regions of variable domains arecalled the framework region (FR), as defined herein. The variabledomains of native heavy and light chains each comprise four FRs (FR1,FR2, FR3 and FR4, respectively), largely adopting a [beta]-sheetconfiguration, connected by the three hypervariable loops. Thehypervariable loops in each chain are held together in close proximityby the FRs and, with the hypervariable loops from the other chain,contribute to the formation of the antigen-binding site of antibodies.Structural analysis of antibodies revealed the relationship between thesequence and the shape of the binding site formed by the complementaritydetermining regions (Chothia et al., 1992. J. Mol. Biol. 227(3):799-817;Tramontano et al., 1990. J. Mol. Biol. 215(1):175-182). Despite theirhigh sequence variability, five of the six loops adopt just a smallrepertoire of main-chain conformations, called “canonical structures”.These conformations are first of all determined by the length of theloops and secondly by the presence of key residues at certain positionsin the loops and in the framework regions that determine theconformation through their packing, hydrogen bonding or the ability toassume unusual main-chain conformations.

“Humanized”—As used herein, the term “humanized” refers to chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from a murine immunoglobulin. Forexample, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary-determining region(CDR) of the recipient are replaced by residues from a CDR of theoriginal antibody (donor antibody) while maintaining the desiredspecificity, affinity, and capacity of the original antibody.

“Humanizing substitutions”—As used herein, the term “humanizingsubstitutions” refers to amino acid substitutions in which the aminoacid residue present at a particular position in the VH or VL domain ofa non-human anti-GPVI antibody (for example a murine anti-GPVI antibody)is replaced with an amino acid residue which occurs at an equivalentposition in a reference human VH or VL domain. The reference human VH orVL domain may be a VH or VL domain encoded by the human germline, inwhich case the substituted residues may be referred to as “germliningsubstitutions”. Humanizing/germlining substitutions may be made in theframework regions and/or the CDRs of an anti-GPVI antibody, definedherein.

“High human homology”—An antibody comprising a heavy chain variabledomain (VH) and a light chain variable domain (VL) will be considered ashaving high human homology if the VH domains and the VL domains, takentogether, exhibit at least 70, 75, 80, 85, 90, 95% or more amino acidsequence identity to the closest matching human germline VH and VLsequences. Antibodies having high human homology may include antibodiescomprising VH and VL domains of native non-human antibodies whichexhibit sufficiently high % sequence identity human germline sequences,as well as engineered, especially humanized, variants of such antibodiesand also “fully human” antibodies. In an embodiment the VH domain of theantibody with high human homology may exhibit an amino acid sequenceidentity or sequence homology of 75%, 80% or greater with one or morehuman VH domains across the framework regions FR1, FR2, FR3 and FR4. Inother embodiments the amino acid sequence identity or sequence homologybetween the VH domain of the protein of the invention and the closestmatching human germline VH domain sequence may be 85% or greater, 90% orgreater, 95% or greater, 97% or greater, or up to 99% or even 100%. Inan embodiment the VH domain of the antibody with high human homology maycontain one or more (e.g., 1 to 20) amino acid sequence mismatchesacross the framework regions FR1, FR2, FR3 and FR4, in comparison to theclosest matched human VH sequence. In another embodiment the VL domainof the antibody with high human homology may exhibit a sequence identityor sequence homology of 80% or greater with one or more human VL domainsacross the framework regions FR1, FR2, FR3 and FR4. In other embodimentsthe amino acid sequence identity or sequence homology between the VLdomain of the protein of the invention and the closest matching humangermline VL domain sequence may be 85% or greater 90% or greater, 95% orgreater, 97% or greater, or up to 99% or even 100%.

In an embodiment the VL domain of the antibody with high human homologymay contain one or more (e.g., 1 to 20, preferably 1 to 10 and morepreferably 1 to 5) amino acid sequence mismatches across the frameworkregions FR1, FR2, FR3 and FR4, in comparison to the closest matchedhuman VL sequence. Before analyzing the percentage sequence identitybetween the antibody with high human homology and human germline VH andVL, the canonical folds may be determined, which allow theidentification of the family of human germline segments with theidentical combination of canonical folds for H1 and H2 or L1 and L2 (andL3). Subsequently the human germline family member that has the highestdegree of sequence homology with the variable region of the antibody ofinterest is chosen for scoring the sequence homology. The determinationof Chothia canonical classes of hypervariable loops L1, L2, L3, H1 andH2 can be performed with the bioinformatics tools publicly available onwebpage www.bioinf.org.uk/abs/chothia.html.page. The output of theprogram shows the key residue requirements in a data file. In these datafiles, the key residue positions are shown with the allowed amino acidsat each position. The sequence of the variable region of the antibody ofinterest is given as input and is first aligned with a consensusantibody sequence to assign the Kabat/Chothia numbering scheme. Theanalysis of the canonical folds uses a set of key residue templatesderived by an automated method developed by Martin and Thornton (Martinet al., 1996. J. Mol. Biol. 263(5):800-815). With the particular humangermline V segment known, which uses the same combination of canonicalfolds for H1 and H2 or L1 and L2 (and L3), the best matching familymember in terms of sequence homology can be determined. Withbioinformatics tools the percentage sequence identity between the VH andVL domain framework amino acid sequences of the antibody of interest andcorresponding sequences encoded by the human germline can be determined,but actually manual alignment of the sequences can be applied as well.Human immunoglobulin sequences can be identified from several proteindata bases, such as VBase (http://vbase.mrc-cpe.cam.ac.uk/) or thePluckthun/Honegger database(http://www.bioc.unizh.ch/antibody/Sequences/Germlines). To compare thehuman sequences to the V regions of VH or VL domains in an antibody ofinterest a sequence alignment algorithm such as available via websiteslike www.expasy.ch/tools/#align can be used, but also manual alignmentwith the limited set of sequences can be performed. Human germline lightand heavy chain sequences of the families with the same combinations ofcanonical folds and with the highest degree of homology with theframework regions 1, 2, and 3 of each chain are selected and comparedwith the variable region of interest; also the FR4 is checked againstthe human germline JH and JK or JL regions. Note that in the calculationof overall percent sequence homology the residues of FR1, FR2 and FR3are evaluated using the closest match sequence from the human germlinefamily with the identical combination of canonical folds. Only residuesdifferent from the closest match or other members of the same familywith the same combination of canonical folds are scored (NB—excludingany primer-encoded differences). However, for the purposes ofhumanization, residues in framework regions identical to members ofother human germline families, which do not have the same combination ofcanonical folds, can be considered “human”, despite the fact that theseare scored “negative” according to the stringent conditions describedabove. This assumption is based on the “mix and match” approach forhumanization, in which each of FR1, FR2, FR3 and FR4 is separatelycompared to its closest matching human germline sequence and thehumanized molecule therefore contains a combination of different FRs aswas done by Qu and colleagues (Qu et al., Clin. Cancer Res. 5:3095-3100(1999)) and Ono and colleagues (Ono et al., Mol. Immunol. 36:387-395(1999)). The boundaries of the individual framework regions may beassigned using the IMGT numbering scheme, which is an adaptation of thenumbering scheme of Chothia (Lefranc et al., Nucleic acid res 27:209-212 (1999); http://im.gt.cines.fr). Antibodies with high humanhomology may comprise hypervariable loops or CDRs having human orhuman-like canonical folds, as discussed in detail below. In anembodiment at least one hypervariable loop or CDR in either the VHdomain or the VL domain of the antibody with high human homology may beobtained or derived from a VH or VL domain of a non-human antibody, yetexhibit a predicted or actual canonical fold structure which issubstantially identical to a canonical fold structure which occurs inhuman antibodies. It is well established in the art that although theprimary amino acid sequences of hypervariable loops present in both VHdomains and VL domains encoded by the human germline are, by definition,highly variable, all hypervariable loops, except CDR H3 of the VHdomain, adopt only a few distinct structural conformations, termedcanonical folds (Chothia et al., J. Mol. Biol. 196:901-917 (1987);Tramontano et al., Proteins 6:382-94 (1989)), which depend on both thelength of the hypervariable loop and presence of the so-called canonicalamino acid residues (Chothia et al., J. Mol. Biol. 196:901-917 (1987)).Actual canonical structures of the hypervariable loops in intact VH orVL domains can be determined by structural analysis (e.g., X-raycrystallography), but it is also possible to predict canonical structureon the basis of key amino acid residues which are characteristic of aparticular structure (discussed further below). In essence, the specificpattern of residues that determines each canonical structure forms a“signature” which enables the canonical structure to be recognized inhypervariable loops of a VH or VL domain of unknown structure; canonicalstructures can therefore be predicted on the basis of primary amino acidsequence alone. The predicted canonical fold structures for thehypervariable loops of any given VH or VL sequence in an antibody withhigh human homology can be analyzed using algorithms which are publiclyavailable from www.bioinf.org.uk/abs/chothia.html,www.biochem.ucl.ac.uk/˜martin/antibodies.html andwww.bioc.unizh.ch/antibody/Sequences/GermlinesNbase_hVk.html. Thesetools permit query VH or VL sequences to be aligned against human VH orVL domain sequences of known canonical structure, and a prediction ofcanonical structure made for the hypervariable loops of the querysequence. In the case of the VH domain, H1 and H2 loops may be scored ashaving a canonical fold structure “substantially identical” to acanonical fold structure known to occur in human antibodies if at leastthe first, and preferable both, of the following criteria are fulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human canonical structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human H1 and H2canonical structural classes (note for the purposes of the foregoinganalysis the H1 and H2 loops are treated separately and each comparedagainst its closest matching human canonical structural class). Theforegoing analysis relies on prediction of the canonical structure ofthe H1 and H2 loops of the antibody of interest. If the actualstructures of the H1 and H2 loops in the antibody of interest are known,for example based on X-ray crystallography, then the H1 and H2 loops inthe antibody of interest may also be scored as having a canonical foldstructure “substantially identical” to a canonical fold structure knownto occur in human antibodies if the length of the loop differs from thatof the closest matching human canonical structural class (typically by+1 or +2 amino acids) but the actual structure of the H1 and H2 loops inthe antibody of interest matches the structure of a human canonicalfold. Key amino acid residues found in the human canonical structuralclasses for the first and second hypervariable loops of human VH domains(H1 and H2) are described by Chothia et al., J. Mol. Biol. 227:799-817(1992), the contents of which are incorporated herein in their entiretyby reference. In particular, Table 3 on page 802 of Chothia et al.,which is specifically incorporated herein by reference, lists preferredamino acid residues at key sites for H1 canonical structures found inthe human germline, whereas Table 4 on page 803, also specificallyincorporated by reference, lists preferred amino acid residues at keysites for CDR H2 canonical structures found in the human germline. In anembodiment, both HI and H2 in the VH domain of the antibody with highhuman homology exhibit a predicted or actual canonical fold structurewhich is substantially identical to a canonical fold structure whichoccurs in human antibodies. Antibodies with high human homology maycomprise a VH domain in which the hypervariable loops H1 and H2 form acombination of canonical fold structures which is identical to acombination of canonical structures known to occur in at least one humangermline VH domain. It has been observed that only certain combinationsof canonical fold structures at H1 and H2 actually occur in VH domainsencoded by the human germline. In an embodiment H1 and H2 in the VHdomain of the antibody with high human homology may be obtained from aVH domain of a non-human species, yet form a combination of predicted oractual canonical fold structures which is identical to a combination ofcanonical fold structures known to occur in a human germline orsomatically mutated VH domain. In non-limiting embodiments H1 and H2 inthe VH domain of the antibody with high human homology may be obtainedfrom a VH domain of a non-human species, and form one of the followingcanonical fold combinations: 1-1, 1-2, 1-3, 1-6, 1-4, 2-1, 3-1 and 3-5.An antibody with high human homology may contain a VH domain whichexhibits both high sequence identity/sequence homology with human VH,and which contains hypervariable loops exhibiting structural homologywith human VH. It may be advantageous for the canonical folds present atH1 and H2 in the VH domain of the antibody with high human homology, andthe combination thereof, to be “correct” for the human VH germlinesequence which represents the closest match with the VH domain of theantibody with high human homology in terms of overall primary amino acidsequence identity. By way of example, if the closest sequence match iswith a human germline VH3 domain, then it may be advantageous for H1 andH2 to form a combination of canonical folds which also occurs naturallyin a human VH3 domain. This may be particularly important in the case ofantibodies with high human homology which are derived from non-humanspecies, e.g., antibodies containing VH and VL domains which are derivedfrom camelid conventional antibodies, especially antibodies containinghumanized camelid VH and VL domains. Thus, in an embodiment the VHdomain of the anti-GPVI antibody with high human homology may exhibit asequence identity or sequence homology of 70% or greater, 80% orgreater, 85% or greater, 90% or greater, 95% or greater, 97% or greater,or up to 99% or even 100% with a human VH domain across the frameworkregions FR1, FR2, FR3 and FR4, and in addition H1 and H2 in the sameantibody are obtained from a non-human VH domain, but form a combinationof predicted or actual canonical fold structures which is the same as acanonical fold combination known to occur naturally in the same human VHdomain. In other embodiments, L1 and L2 in the VL domain of the antibodywith high human homology are each obtained from a VL domain of anon-human species, and each exhibits a predicted or actual canonicalfold structure which is substantially identical to a canonical foldstructure which occurs in human antibodies. As with the VH domains, thehypervariable loops of VL domains of both VLambda and VKappa types canadopt a limited number of conformations or canonical structures,determined in part by length and also by the presence of key amino acidresidues at certain canonical positions. Within an antibody of interesthaving high human homology, L1, L2 and L3 loops obtained from a VLdomain of a non-human species, e.g., a Camelidae species, may be scoredas having a canonical fold structure “substantially identical” to acanonical fold structure known to occur in human antibodies if at leastthe first, and preferable both, of the following criteria are fulfilled:1. An identical length, determined by the number of residues, to theclosest matching human structural class.2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human L1 or L2canonical structural classes, from either the VLambda or the VKapparepertoire (note for the purposes of the foregoing analysis the L1 andL2 loops are treated separately and each compared against its closestmatching human canonical structural class). The foregoing analysisrelies on prediction of the canonical structure of the L1, L2 and L3loops in the VL domain of the antibody of interest. If the actualstructure of the L1, L2 and L3 loops is known, for example based onX-ray crystallography, then L1, L2 or L3 loops derived from the antibodyof interest may also be scored as having a canonical fold structure“substantially identical” to a canonical fold structure known to occurin human antibodies if the length of the loop differs from that of theclosest matching human canonical structural class (typically by +1 or +2amino acids) but the actual structure of the loops in the antibody ofinterest matches a human canonical fold. Key amino acid residues foundin the human canonical structural classes for the CDRs of human VLambdaand VKappa domains are described by Morea et al., Methods, 20: 267-279(2000) and Martin et al., J. Mol. Biol., 263:800-815 (1996). Thestructural repertoire of the human VKappa domain is also described byTomlinson et al., EMBO J. 14:4628-4638 (1995), and that of the VLambdadomain by Williams et al., J. Mol. Biol., 264:220-232 (1996). Thecontents of all these documents are to be incorporated herein byreference. L1 and L2 in the VL domain of an antibody with high humanhomology may form a combination of predicted or actual canonical foldstructures which is identical to a combination of canonical foldstructures known to occur in a human germline VL domain. In non-limitingembodiments LI and L2 in the VLambda domain of an antibody with highhuman homology may form one of the following canonical foldcombinations: 11-7, 13-7(A,B,C), 14-7(A,B), 12-11, 14-11 and 12-12 (asdefined in Williams et al., J. Mol. Biol. 264:220-32 (1996) and as shownon http://www.bioc.uzh.ch/antibody/Sequences/GermlinesNBase_hVL.html).In non-limiting embodiments L1 and L2 in the VKappa domain may form oneof the following canonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (asdefined in Tomlinson et al., EMBO J. 14:4628-38 (1995) and as shown onhttp://www.bioc.uzh.ch/antibody/Sequences/GermlinesNBase_hVK.html).

In a further embodiment, all three of L1, L2 and L3 in the VL domain ofan antibody with high human homology may exhibit a substantially humanstructure. It is preferred that the VL domain of the antibody with highhuman homology exhibit both high sequence identity/sequence homologywith human VL, and also that the hypervariable loops in the VL domainexhibit structural homology with human VL.

In an embodiment, the VL domain of the anti-GPVI antibody with highhuman homology may exhibit a sequence identity of 70% or greater, 80% orgreater, 85% or greater, 90% or greater, 95% or greater, 97% or greater,or up to 99% or even 100% with a human VL domain across the frameworkregions FR1, FR2, FR3 and FR4, and in addition hypervariable loop L1 andhypervariable loop L2 may form a combination of predicted or actualcanonical fold structures which is the same as a canonical foldcombination known to occur naturally in the same human VL domain. It is,of course, envisaged that VH domains exhibiting high sequenceidentity/sequence homology with human VH, and also structural homologywith hypervariable loops of human VH will be combined with VL domainsexhibiting high sequence identity/sequence homology with human VL, andalso structural homology with hypervariable loops of human VL to provideantibodies with high human homology containing VH/VL pairings withmaximal sequence and structural homology to human-encoded VH/VLpairings.

“Immunospecific”, “specific for” or to “specifically bind”—As usedherein, an antibody is said to be “immunospecific”, “specific for” or to“specifically bind” an antigen if it reacts at a detectable level withthe antigen, preferably with an affinity constant, K_(A), of greaterthan or equal to about 10⁶ M⁻¹, greater than or equal to about 10⁷ M⁻¹,or greater than or equal to 10⁸ M⁻¹, or greater than or equal to 1.510⁸M⁻¹, or greater than or equal to 10⁹ M⁻¹ or greater than or equal to5 10⁹ M⁻¹. Affinity of an antibody for its cognate antigen is alsocommonly expressed as a dissociation constant K_(D), and in certainembodiments, an antibody specifically binds to antigen if it binds witha K_(D) of less than or equal to 10⁻⁶ M, less than or equal to 10⁻⁷ M,or less than or equal to 1.5 10⁻⁸ M, or less than or equal to 10⁻⁸ M, orless than or equal to 5 10⁻⁹ M or less than or equal to 10⁻⁹ M.Affinities of antibodies can be readily determined using conventionaltechniques, for example, those described by Scatchard G et al. (Theattractions of proteins for small molecules and ions. Ann NY Acad Sci1949; 51: 660-672). Binding properties of an antibody to antigens, cellsor tissues thereof may generally be determined and assessed usingimmunodetection methods including, for example, ELISA,immunofluorescence-based assays, such as immuno-histochemistry (IHC)and/or fluorescence-activated cell sorting (FACS) or by surface plasmonresonance (SPR, BIAcore).

“Isolated nucleic acid”—As used herein, an “isolated nucleic acid” is anucleic acid that is substantially separated from other genome DNAsequences as well as proteins or complexes such as ribosomes andpolymerases, which naturally accompany a native sequence. The termembraces a nucleic acid sequence that has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates and chemically synthesized analogues or analogues biologicallysynthesized by heterologous systems. A substantially pure nucleic acidincludes isolated forms of the nucleic acid. Of course, this refers tothe nucleic acid as originally isolated and does not exclude genes orsequences later added to the isolated nucleic acid by the hand of man.

The term “polypeptide” is used in its conventional meaning, i.e., as asequence of less than 100 amino acids. A polypeptide usually refers to amonomeric entity. The term “protein” refers to a sequence of more than100 amino acids and/or to a multimeric entity. The proteins of theinvention are not limited to a specific length of the product.

This term does not refer to or exclude post-expression modifications ofthe protein, for example, glycosylation, acetylation, phosphorylationand the like, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring. A protein may be anentire protein, or a subsequence thereof. Particular proteins ofinterest in the context of this invention are amino acid subsequencescomprising CDRs and being capable of binding an antigen. An “isolatedprotein” is one that has been identified and separated and/or recoveredfrom a component of its natural environment. In preferred embodiments,the isolated protein will be purified (1) to greater than 80, 85, 90,95% by weight of protein as determined by the Lowry method, and mostpreferably more than 96, 97, 98, or 99% by weight, (2) to a degreesufficient to obtain at least residues of N-terminal or internal aminoacid sequence by use of a spinning cup sequenator, or (3) to homogeneityby SDS-PAGE under reducing or non-reducing conditions using Coomassieblue or, preferably, silver staining. Isolated protein includes theprotein in situ within recombinant cells since at least one component ofthe protein's natural environment will not be present. Ordinarily,however, isolated protein will be prepared by at least one purificationstep.

“Identity” or “identical”—As used herein, the term “identity” or“identical”, when used in a relationship between the sequences of two ormore amino acid sequences, refers to the degree of sequence relatednessbetween amino acid sequences, as determined by the number of matchesbetween strings of two or more amino acid residues. “Identity” measuresthe percent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated amino acid sequences can be readily calculated by known methods.Such methods include, but are not limited to, those described in ArthurM. Lesk, Computational Molecular Biology: Sources and Methods forSequence Analysis (New-York: Oxford University Press, 1988); Douglas W.Smith, Biocomputing: Informatics and Genome Projects (New-York: AcademicPress, 1993); Hugh G. Griffin and Annette M. Griffin, Computer Analysisof Sequence Data, Part 1 (New Jersey: Humana Press, 1994); Gunnar vonHeinje, Sequence Analysis in Molecular Biology: Treasure Trove orTrivial Pursuit (Academic Press, 1987); Michael Gribskov and JohnDevereux, Sequence Analysis Primer (New York: M. Stockton Press, 1991);and Carillo et al., 1988. SIAM J. Appl. Math. 48(5):1073-1082. Preferredmethods for determining identity are designed to give the largest matchbetween the sequences tested. Methods of determining identity aredescribed in publicly available computer programs. Preferred computerprogram methods for determining identity between two sequences includethe GCG program package, including GAP (Devereux et al., 1984. Nucl.Acid. Res. 12(1 Pt 1):387-395; Genetics Computer Group, University ofWisconsin Biotechnology Center, Madison, Wis.), BLASTP, BLASTN, TBLASTNand FASTA (Altschul et al., 1990. J. Mol. Biol. 215(3):403-410). TheBLASTX program is publicly available from the National Center forBiotechnology Information (NCBI) and other sources (BLAST Manual,Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990.J. Mol. Biol. 215(3):403-410). The well-known Smith Waterman algorithmmay also be used to determine identity.

“Modified antibody”—As used herein, the term “modified antibody”includes synthetic forms of antibodies which are altered such that theyare not naturally occurring, e.g., antibodies that comprise at least twoheavy chain regions but not two complete heavy chains (such as, domaindeleted antibodies or minibodies); multispecific forms of antibodies(e.g., bispecific, trispecific, etc.) altered to bind to two or moredifferent antigens or to different epitopes on a single antigen; heavychain molecules joined to scFv molecules and the like. ScFv moleculesare known in the art and are described, e.g., in U.S. Pat. No.5,892,019. In addition, the term “modified antibody” includesmultivalent forms of antibodies (e.g., trivalent, tetravalent, etc.,antibodies that bind to three or more copies of the same antigen). Inanother embodiment, a modified antibody of the invention is a fusionprotein comprising at least one heavy chain region lacking a CH2 domainand comprising a binding domain of a protein comprising the bindingregion of one member of a receptor ligand pair.

“Mammal”—As used herein, the term “mammal” refers to any mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is a primate, more preferably ahuman.

“Monoclonal antibody”—As used herein, the term “monoclonal antibody”refers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprised in thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to polyclonal antibody preparations thatinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they may be synthesizeduncontaminated by other antibodies. The modifier “monoclonal” is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies useful in the presentinvention may be prepared by the hybridoma methodology first describedby Kohler et al., Nature, 256:495 (1975), or may be made usingrecombinant DNA methods in bacterial, eukaryotic animal or plant cells(see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” mayalso be isolated from phage antibody libraries using the techniquesdescribed in Clackson et al., Nature, 352:624-628 (1991) and Marks etal., J. Mol. Biol., 222:581-597 (1991), for example.

“Native sequence”—As used herein, the term “native sequence” nucleotiderefers to a polynucleotide that has the same nucleotide sequence as apolynucleotide derived from nature. Accordingly, a “native sequence”protein is one that has the same amino acid sequence as a protein (e.g.,antibody) derived from nature (e.g., from any species). Such nativesequence polynucleotides and proteins can be isolated from nature or canbe produced by recombinant or synthetic means. A polynucleotide“variant”, as the term is used herein, is a polynucleotide thattypically differs from a polynucleotide specifically disclosed herein inone or more substitutions, deletions, additions and/or insertions. Suchvariants may be naturally occurring or may be synthetically generated,for example, by modifying one or more of the polynucleotide sequences asdescribed herein and evaluating one or more biological activities of theencoded proteins as described herein and/or using any of a number oftechniques well known in the art. A protein “variant”, as the term isused herein, is a protein that typically differs from a proteinspecifically disclosed herein in one or more substitutions, deletions,additions and/or insertions. Such variants may be naturally occurring ormay be synthetically generated, for example, by modifying one or more ofthe above protein sequences and evaluating one or more biologicalactivities of the protein as described herein and/or using any of anumber of techniques well known in the art. Modifications may be made inthe structure of the polynucleotides and proteins of the presentinvention and still obtain a functional molecule that encodes a variantor derivative protein with desirable characteristics. When it is desiredto alter the amino acid sequence of a protein to create an equivalent,or even an improved, variant or region of a protein as described herein,one skilled in the art will typically change one or more of the codonsof the encoding DNA sequence. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of its ability to bind other proteins (e.g., antigens)or cells. Since it is the binding capacity and nature of a protein thatdefines that protein's biological functional activity, certain aminoacid sequence substitutions can be made in a protein sequence, and ofcourse, its underlying DNA coding sequence, and nevertheless obtain aprotein with similar properties. It is thus contemplated that variouschanges may be made in the amino acid sequences of the disclosedcompositions, or corresponding DNA sequences that encode said proteinswithout appreciable loss of their biological utility or activity. Inmany instances, a protein variant will contain one or more conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the protein to besubstantially unchanged. As outlined above, amino acid substitutions aregenerally therefore based on the relative similarity of the amino acidside-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. Exemplary substitutions thattake several of the foregoing characteristics into consideration arewell known to those of skill in the art and include: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine. Amino acid substitutionsmay further be made on the basis of similarity in polarity, charge,solubility, hydrophobicity, hydrophilicity and/or the amphipathic natureof the residues. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine and arginine; and amino acids with uncharged polar head groupshaving similar hydrophilicity values include leucine, isoleucine andvaline; glycine and alanine; asparagine and glutamine; and serine,threonine, phenylalanine and tyrosine. Other groups of amino acids thatmay represent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain non-conservative changes. In a preferredembodiment, variant proteins differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of theprotein.

“Pharmaceutically acceptable excipient”—As used herein, the term“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. Said excipientdoes not produce an adverse, allergic or other untoward reaction whenadministered to an animal, preferably a human. For human administration,preparations should meet sterility, pyrogenicity, and general safety andpurity standards as required by regulatory offices, such as, forexample, FDA Office or EMA.

“Specificity”—As used herein, the term “specificity” refers to theability to specifically bind (e.g., immunoreact with) a given target,e.g., GPVI. A protein may be monospecific and contain one or morebinding sites which specifically bind a target, or a protein may bemultispecific and contain two or more binding sites which specificallybind the same or different targets. In an embodiment, an antibody asdescribed herein is specific for more than one target. For example, inan embodiment, a multispecific binding molecule binds to GPVI and asecond molecule.

“Single-chain Fv” also abbreviated as “sFv” or “scFv”—As used herein,the terms “Single-chain Fv”, “sFv” or “scFv” are antibody fragments thatcomprise the VH and VL antibody domains connected into a single aminoacid chain. Preferably, the sFv amino acid sequence further comprises apeptidic linker between the VH and VL domains that enables the sFv toform the desired structure for antigen binding. For a review of sFv, seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994);Borrebaeck 1995, infra.

“Subject”—As used herein, the term “subject” refers to a mammal,preferably a human. In one embodiment, a subject may be a “patient”,i.e. a warm-blooded animal, more preferably a human, who/which isawaiting the receipt of, or is receiving medical care or was/is/will bethe object of a medical procedure, or is monitored for the developmentof a disease.

“Synthetic”—As used herein, the term “synthetic” with respect toproteins includes proteins which comprise an amino acid sequence that isnot naturally occurring. For example, non-naturally occurring proteinsare modified forms of naturally occurring proteins (e.g., comprising amutation such as an addition, substitution or deletion) or proteinswhich comprise a first amino acid sequence (which may or may not benaturally occurring) that is linked in a linear sequence of amino acidsto a second amino acid sequence (which may or may not be naturallyoccurring) to which it is not naturally linked in nature.

“Therapeutically effective amount” means level or amount of agent thatis aimed at, without causing significant negative or adverse sideeffects to the target, (1) delaying or preventing the onset ofGPVI-related disease; (2) slowing down or stopping the progression,aggravation, or deterioration of one or more symptoms of theGPVI-related disease; (3) bringing about ameliorations of the symptomsof the GPVI-related disease; (4) reducing the severity or incidence ofthe GPVI-related disease; or (5) curing the GPVI-related disease. Atherapeutically effective amount may be administered prior to the onsetof the GPVI-related disease, for a prophylactic or preventive action.Alternatively or additionally, the therapeutically effective amount maybe administered after initiation of the GPVI-related disease, for atherapeutic action.

“Treating” or “treatment” or “alleviation”—As used herein, the terms“treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures; wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for the targeted pathologic condition or disorder if, afterreceiving a therapeutic amount of a protein according to the presentinvention, the subject or mammal shows observable and/or measurableimprovement in one or more of the following: reduction in the number ofpathogenic cells; reduction in the percent of total cells that arepathogenic; and/or relief to some extent, of one or more of the symptomsassociated with the specific disease or condition; reduced morbidity andmortality, and/or improvement in quality of life issues. The aboveparameters for assessing successful treatment and improvement in thedisease are readily measurable by routine procedures familiar to aphysician.

“Variable region” or “variable domain”—As used herein, the term“variable” refers to the fact that certain regions of the variabledomains VH and VL differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits target antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called “hypervariable loops” in each of the VL domain andthe VH domain which form part of the antigen binding site. The first,second and third hypervariable loops of the VLambda light chain domainare referred to herein as L1 (λ), L2 (λ) and L3 (λ) and may be definedas comprising residues 24-33 (L1(λ), consisting of 9, 10 or 11 aminoacid residues), 49-53 L2 (λ), consisting of 3 residues) and 90-96(L3(λ), consisting of 6 residues) in the VL domain (Morea et al.,Methods 20:267-279 (2000)). The first, second and third hypervariableloops of the VKappa light chain domain are referred to herein as L1(κ),L2(κ) and L3(κ) and may be defined as comprising residues 25-33 (L1(κ),consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(κ), consistingof 3 residues) and 90-97 (L3(κ), consisting of 6 residues) in the VLdomain (Morea et al., Methods 20:267-279 (2000)). The first, second andthird hypervariable loops of the VH domain are referred to herein as H1,H2 and H3 and may be defined as comprising residues 25-33 (HI,consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4residues) and 91-105 (H3, highly variable in length) in the VH domain(Morea et al., Methods 20:267-279 (2000)). Unless otherwise indicated,the terms L1, L2 and L3 respectively refer to the first, second andthird hypervariable loops of a VL domain, and encompass hypervariableloops obtained from both VKappa and VLambda isotypes. The terms H1, H2and H3 respectively refer to the first, second and third hypervariableloops of the VH domain, and encompass hypervariable loops obtained fromany of the known heavy chain isotypes, including [gamma], [epsilon],[delta], [alpha] or [mu]. The hypervariable loops L1, L2, L3, H1, H2 andH3 may each comprise part of a “complementarity determining region” or“CDR”, as defined hereinabove.

“Valency”—As used herein, the term “valency” refers to the number ofpotential target binding sites in a protein. Each target binding sitespecifically binds one target molecule or specific site on a targetmolecule. When a protein comprises more than one target binding site,each target binding site may specifically bind the same or differentmolecules (e.g., may bind to different ligands or different antigens, ordifferent epitopes on the same antigen). The subject binding moleculespreferably have at least one binding site specific for a human GPVImolecule. Preferably, the proteins provided herein are monovalent.

An object of the present invention is an isolated humanized proteinbinding to human Glycoprotein VI (hGPVI) for treating or for use intreating a GPVI-related condition in a subject in need thereof, whereinsaid isolated humanized protein is administered (or is to beadministered) during at least 2 hours to the subject, preferably duringat least 4 to 6 hours.

In one embodiment, the isolated humanized proteins binding to human GPVIare isolated humanized antibodies against human GPVI.

In one embodiment, the isolated protein is purified.

In one embodiment, the protein binds to the extracellular domain ofGPVI.

In one embodiment, the protein binds to the Ig-like C2-type domain 2(D2) of human GPVI.

Thus, in one embodiment, the protein binds to an epitope comprising atleast one amino acid residue from amino acid residues 114 to 207 ofhuman GPVI (SEQ ID NO: 13) or from a sequence sharing at least 60%, 70%,75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acidresidues 114 to 207 of human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope comprises at least one amino acidresidue from amino acid residues 114 to 187, preferably from 115 to 187,more preferably from 116 to 187, more preferably from 117 to 187, morepreferably from 118 to 186, more preferably from 119 to 185, morepreferably from 120 to 184, even more preferably from 121 to 183 ofhuman GPVI (SEQ ID NO: 13), or from a sequence sharing at least 60%,70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acidresidues 114 to 187, preferably from 115 to 187, more preferably from116 to 187, more preferably from 117 to 187, more preferably from 118 to186, more preferably from 119 to 185, more preferably from 120 to 184,even more preferably from 121 to 183 of human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope comprises at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more amino acid residues from amino acidresidues 114 to 187, preferably from 115 to 187, more preferably from116 to 187, more preferably from 117 to 187, more preferably from 118 to186, more preferably from 119 to 185, more preferably from 120 to 184,even more preferably from 121 to 183 of human GPVI (SEQ ID NO: 13), orfrom a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%,98%, 99% of identity over amino acid residues 114 to 187, preferablyfrom 115 to 187, more preferably from 116 to 187, more preferably from117 to 187, more preferably from 118 to 186, more preferably from 119 to185, more preferably from 120 to 184, even more preferably from 121 to183 of human GPVI (SEQ ID NO: 13).

In one embodiment, the epitope comprises at least one (e.g., 1, 2, 3, 4,5, 6, 7, or 8) of the following residues in GPVI sequence (SEQ ID NO:13): 125S, 126S, 128G, 133Q, 136T, 171T, 172A and/or 174H. In oneembodiment, the epitope comprises at least one (e.g., 1, 2, 3, 4, 5, 6,or 7) of the following residues in GPVI sequence (SEQ ID NO: 13): 125S,126S, 128G, 133Q, 171T, 172A and/or 174H.

In one embodiment, said epitope comprises at least one amino acidresidue from amino acid residues 114 to 142, preferably from 115 to 141,more preferably from 116 to 140, more preferably from 117 to 139, morepreferably from 118 to 138, more preferably from 119 to 137, morepreferably from 120 to 136, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13), or from a sequence sharing atleast 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity overamino acid residues 114 to 142, preferably from 115 to 141, morepreferably from 116 to 140, more preferably from 117 to 139, morepreferably from 118 to 138, more preferably from 119 to 137, morepreferably from 120 to 136, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope comprises at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28 amino acid residues from 114 to 142, preferably from 115 to141, more preferably from 116 to 140, more preferably from 117 to 139,more preferably from 118 to 138, more preferably from 119 to 137, morepreferably from 120 to 136, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13), or from a sequence sharing atleast 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity overamino acid residues 114 to 142, preferably from 115 to 141, morepreferably from 116 to 140, more preferably from 117 to 139, morepreferably from 118 to 138, more preferably from 119 to 137, morepreferably from 120 to 136, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope comprises at least one amino acidresidue from amino acid residues 114 to 135, preferably from 115 to 135,more preferably from 116 to 135, more preferably from 117 to 135, morepreferably from 118 to 135, more preferably from 119 to 135, morepreferably from 120 to 135, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13), or from a sequence sharing atleast 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity overamino acid residues 114 to 135, preferably from 115 to 135, morepreferably from 116 to 135, more preferably from 117 to 135, morepreferably from 118 to 135, more preferably from 119 to 135, morepreferably from 120 to 135, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope comprises at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 amino acid residuesfrom 114 to 135, preferably from 115 to 135, more preferably from 116 to135, more preferably from 117 to 135, more preferably from 118 to 135,more preferably from 119 to 135, more preferably from 120 to 135, evenmore preferably from 121 to 135 or from 121 to 136 of human GPVI (SEQ IDNO: 13), or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%,95%, 96%, 97%, 98%, 99% of identity over amino acid residues 114 to 135,preferably from 115 to 135, more preferably from 116 to 135, morepreferably from 117 to 135, more preferably from 118 to 135, morepreferably from 119 to 135, more preferably from 120 to 135, even morepreferably from 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:13).

In one embodiment, the epitope comprises at least one (e.g., 1, 2, 3, 4,or 5) of the following residues in GPVI sequence (SEQ ID NO: 13): 125S,126S, 128G, 133Q, and/or 136T. In one embodiment, the epitope comprisesat least one (e.g., 1, 2, 3, or 4) of the following residues in GPVIsequence (SEQ ID NO: 13): 125S, 126S, 128G, and/or 133Q.

In one embodiment, said epitope comprises at least one amino acidresidue from amino acid residues 165 to 187, preferably from 166 to 186,more preferably from 167 to 185, more preferably from 168 to 184, evenmore preferably from 169 to 183 of human GPVI (SEQ ID NO: 13), or from asequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,99% of identity over amino acid residues 165 to 187, preferably from 166to 186, more preferably from 167 to 185, more preferably from 168 to184, even more preferably from 169 to 183 of human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope comprises at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 amino acidresidues from 165 to 187, preferably from 166 to 186, more preferablyfrom 167 to 185, more preferably from 168 to 184, even more preferablyfrom 169 to 183 of human GPVI (SEQ ID NO: 13), or from a sequencesharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% ofidentity over amino acid residues 165 to 187, preferably from 166 to186, more preferably from 167 to 185, more preferably from 168 to 184,even more preferably from 169 to 183 of human GPVI (SEQ ID NO: 13).

In one embodiment, the epitope comprises at least one (e.g. 1, 2 or 3)of the following residues in GPVI sequence: 171T, 172A and/or 174H.

In one embodiment, the isolated protein binds to a conformationalepitope.

In one embodiment, said conformational epitope comprises at least one,preferably at least two, amino acid residue(s) of human GPVI.

In one embodiment, said conformational epitope comprises at least one,preferably at least two, amino acid residue(s) of the Ig-like C2-typedomain 2 (D2) of human GPVI.

In one embodiment, said conformational epitope comprises at least one,preferably at least two, amino acid residue(s) from 114 to 207 of humanGPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least oneamino acid residue from amino acid residues 114 to 187, preferably from115 to 187, more preferably from 116 to 187, more preferably from 117 to187, more preferably from 118 to 186, more preferably from 119 to 185,more preferably from 120 to 184, even more preferably from 121 to 183 ofhuman GPVI (SEQ ID NO: 13), or from a sequence sharing at least 60%,70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acidresidues 114 to 187, preferably from 115 to 187, more preferably from116 to 187, more preferably from 117 to 187, more preferably from 118 to186, more preferably from 119 to 185, more preferably from 120 to 184,even more preferably from 121 to 183 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues fromamino acid residues 114 to 187, preferably from 115 to 187, morepreferably from 116 to 187, more preferably from 117 to 187, morepreferably from 118 to 186, more preferably from 119 to 185, morepreferably from 120 to 184, even more preferably from 121 to 183 ofhuman GPVI (SEQ ID NO: 13), or from a sequence sharing at least 60%,70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acidresidues 114 to 187, preferably from 115 to 187, more preferably from116 to 187, more preferably from 117 to 187, more preferably from 118 to186, more preferably from 119 to 185, more preferably from 120 to 184,even more preferably from 121 to 183 of human GPVI (SEQ ID NO: 13).

In one embodiment, the conformational epitope comprises at least one(e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following residues in GPVIsequence (SEQ ID NO: 13): 125S, 126S, 128G, 133Q, 136T, 171T, 172Aand/or 174H. In one embodiment, the conformational epitope comprises atleast one (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following residues inGPVI sequence (SEQ ID NO: 13): 125S, 126S, 128G, 133Q, 171T, 172A and/or174H.

In one embodiment, said conformational epitope comprises at least oneamino acid residue from amino acid residues 114 to 142, preferably from115 to 141, more preferably from 116 to 140, more preferably from 117 to139, more preferably from 118 to 138, more preferably from 119 to 137,more preferably from 120 to 136, even more preferably from 121 to 135 orfrom 121 to 136 of human GPVI (SEQ ID NO: 13), or from a sequencesharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% ofidentity over amino acid residues 114 to 142, preferably from 115 to141, more preferably from 116 to 140, more preferably from 117 to 139,more preferably from 118 to 138, more preferably from 119 to 137, morepreferably from 120 to 136, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28 amino acid residues from amino acid residues 114to 142, preferably from 115 to 141, more preferably from 116 to 140,more preferably from 117 to 139, more preferably from 118 to 138, morepreferably from 119 to 137, more preferably from 120 to 136, even morepreferably from 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:13), or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%,96%, 97%, 98%, 99% of identity over amino acid residues 114 to 142,preferably from 115 to 141, more preferably from 116 to 140, morepreferably from 117 to 139, more preferably from 118 to 138, morepreferably from 119 to 137, more preferably from 120 to 136, even morepreferably from 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:13).

In one embodiment, said conformational epitope comprises at least oneamino acid residue from amino acid residues 114 to 135, preferably from115 to 135, more preferably from 116 to 135, more preferably from 117 to135, more preferably from 118 to 135, more preferably from 119 to 135,more preferably from 120 to 135, even more preferably from 121 to 135 orfrom 121 to 136 of human GPVI (SEQ ID NO: 13), or from a sequencesharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% ofidentity over amino acid residues 114 to 135, preferably from 115 to135, more preferably from 116 to 135, more preferably from 117 to 135,more preferably from 118 to 135, more preferably from 119 to 135, morepreferably from 120 to 135, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21amino acid residues from amino acid residues 114 to 135, preferably from115 to 135, more preferably from 116 to 135, more preferably from 117 to135, more preferably from 118 to 135, more preferably from 119 to 135,more preferably from 120 to 135, even more preferably from 121 to 135 orfrom 121 to 136 of human GPVI (SEQ ID NO: 13), or from a sequencesharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% ofidentity over amino acid residues 114 to 135, preferably from 115 to135, more preferably from 116 to 135, more preferably from 117 to 135,more preferably from 118 to 135, more preferably from 119 to 135, morepreferably from 120 to 135, even more preferably from 121 to 135 or from121 to 136 of human GPVI (SEQ ID NO: 13).

In one embodiment, the conformational epitope comprises at least one(e.g., 1, 2, 3, 4, or 5) of the following residues in GPVI sequence (SEQID NO: 13): 125S, 126S, 128G, 133Q, and/or 136T. In one embodiment, theconformational epitope comprises at least one (e.g., 1, 2, 3, or 4) ofthe following residues in GPVI sequence (SEQ ID NO: 13): 125S, 126S,128G, and/or 133Q.

In one embodiment, said conformational epitope comprises at least oneamino acid residue from amino acid residues 165 to 187, preferably from166 to 186, more preferably from 167 to 185, more preferably from 168 to184, even more preferably from 169 to 183 of human GPVI (SEQ ID NO: 13)or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%,97%, 98%, 99% of identity over amino acid residues 165 to 187,preferably from 166 to 186, more preferably from 167 to 185, morepreferably from 168 to 184, even more preferably from 169 to 183 ofhuman GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22amino acid residues from amino acid residues 165 to 187, preferably from166 to 186, more preferably from 167 to 185, more preferably from 168 to184, even more preferably from 169 to 183 of human GPVI (SEQ ID NO: 13)or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%,97%, 98%, 99% of identity over amino acid residues 165 to 187,preferably from 166 to 186, more preferably from 167 to 185, morepreferably from 168 to 184, even more preferably from 169 to 183 ofhuman GPVI (SEQ ID NO: 13).

In one embodiment, the conformational epitope comprises at least one(e.g. 1, 2 or 3) of the following residues in GPVI sequence: 171T, 172Aand/or 174H.

In one embodiment, said conformational epitope comprises:

-   -   at least one amino acid residue from amino acid residues 114 to        142, preferably from 115 to 141, more preferably from 116 to        140, more preferably from 117 to 139, more preferably from 118        to 138, more preferably from 119 to 137, more preferably from        120 to 136, even more preferably from 121 to 135 or from 121 to        136 of human GPVI (SEQ ID NO: 13) or from a sequence sharing at        least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of        identity over amino acid residues 114 to 142, preferably from        115 to 141, more preferably from 116 to 140, more preferably        from 117 to 139, more preferably from 118 to 138, more        preferably from 119 to 137, more preferably from 120 to 136,        even more preferably from 121 to 135 or from 121 to 136 of human        GPVI (SEQ ID NO: 13); and    -   at least one amino acid residue from amino acid residues 165 to        187, preferably from 166 to 186, more preferably from 167 to        185, more preferably from 168 to 184, even more preferably from        169 to 183 of human GPVI (SEQ ID NO: 13) or from a sequence        sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,        99% of identity over amino acid residues 165 to 187, preferably        from 166 to 186, more preferably from 167 to 185, more        preferably from 168 to 184, even more preferably from 169 to 183        of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises:

-   -   at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 amino acid        residues from amino acid residues 114 to 142, preferably from        115 to 141, more preferably from 116 to 140, more preferably        from 117 to 139, more preferably from 118 to 138, more        preferably from 119 to 137, more preferably from 120 to 136,        even more preferably from 121 to 135 or from 121 to 136 of human        GPVI (SEQ ID NO: 13) or from a sequence sharing at least 60%,        70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over        amino acid residues 114 to 142, preferably from 115 to 141, more        preferably from 116 to 140, more preferably from 117 to 139,        more preferably from 118 to 138, more preferably from 119 to        137, more preferably from 120 to 136, even more preferably from        121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO: 13); and    -   at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22 amino acid residues from amino acid        residues 165 to 187, preferably from 166 to 186, more preferably        from 167 to 185, more preferably from 168 to 184, even more        preferably from 169 to 183 of human GPVI (SEQ ID NO: 13) or from        a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%,        97%, 98%, 99% of identity over amino acid residues 165 to 187,        preferably from 166 to 186, more preferably from 167 to 185,        more preferably from 168 to 184, even more preferably from 169        to 183 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises:

-   -   at least one amino acid residue from amino acid residues 114 to        135, preferably from 115 to 135, more preferably from 116 to        135, more preferably from 117 to 135, more preferably from 118        to 135, more preferably from 119 to 135, more preferably from        120 to 135, even more preferably from 121 to 135 or from 121 to        136 of human GPVI (SEQ ID NO: 13) or from a sequence sharing at        least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of        identity over amino acid residues 114 to 135, preferably from        115 to 135, more preferably from 116 to 135, more preferably        from 117 to 135, more preferably from 118 to 135, more        preferably from 119 to 135, more preferably from 120 to 135,        even more preferably from 121 to 135 or from 121 to 136 of human        GPVI (SEQ ID NO: 13); and    -   at least one amino acid residue from amino acid residues 165 to        187, preferably from 166 to 186, more preferably from 167 to        185, more preferably from 168 to 184, even more preferably from        169 to 183 of human GPVI (SEQ ID NO: 13) or from a sequence        sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,        99% of identity over amino acid residues 165 to 187, preferably        from 166 to 186, more preferably from 167 to 185, more        preferably from 168 to 184, even more preferably from 169 to 183        of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises:

-   -   at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21 amino acid residues from amino acid residues        114 to 135, preferably from 115 to 135, more preferably from 116        to 135, more preferably from 117 to 135, more preferably from        118 to 135, more preferably from 119 to 135, more preferably        from 120 to 135, even more preferably from 121 to 135 or from        121 to 136 of human GPVI (SEQ ID NO: 13) or from a sequence        sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,        99% of identity over amino acid residues 114 to 135, preferably        from 115 to 135, more preferably from 116 to 135, more        preferably from 117 to 135, more preferably from 118 to 135,        more preferably from 119 to 135, more preferably from 120 to        135, even more preferably from 121 to 135 or from 121 to 136 of        human GPVI (SEQ ID NO: 13); and    -   at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, 21, 22 amino acid residues from amino acid        residues 165 to 187, preferably from 166 to 186, more preferably        from 167 to 185, more preferably from 168 to 184, even more        preferably from 169 to 183 of human GPVI (SEQ ID NO: 13) or from        a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%,        97%, 98%, 99% of identity over amino acid residues 165 to 187,        preferably from 166 to 186, more preferably from 167 to 185,        more preferably from 168 to 184, even more preferably from 169        to 183 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least oneamino acid residue from amino acid residues 121 to 135 or from 121 to136 of human GPVI (SEQ ID NO: 13) or from a sequence sharing at least60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over aminoacid residues 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:13); and at least one amino acid residue from amino acid residues 169 to183 of human GPVI (SEQ ID NO: 13) or from a sequence sharing at least60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over aminoacid residues 169 to 183 of human GPVI (SEQ ID NO: 13).

Thus, in one embodiment, the protein binds to a conformational epitopecomprising at least one amino acid residue from amino acid residues 121to 135 or from 121 to 136 of human GPVI (SEQ ID NO: 13) or from asequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,99% of identity over amino acid residues 121 to 135 or from 121 to 136of human GPVI (SEQ ID NO: 13); and at least one amino acid residue fromamino acid residues 169 to 183 of human GPVI (SEQ ID NO: 13) or from asequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,99% of identity over amino acid residues 169 to 183 of human GPVI (SEQID NO: 13).

In one embodiment, said conformational epitope comprises at least oneamino acid residue from amino acid residues 121 to 135 or from 121 to136 of human GPVI (SEQ ID NO: 13) or from a sequence sharing at least60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over aminoacid residues 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:13); and at least one amino acid residue from amino acid residues 169 to180 of human GPVI (SEQ ID NO: 13) or from a sequence sharing at least60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over aminoacid residues 169 to 180 of human GPVI (SEQ ID NO: 13).

Thus, in one embodiment, the protein binds to a conformational epitopecomprising at least one amino acid residue from amino acid residues 121to 135 or from 121 to 136 of human GPVI (SEQ ID NO: 13) or from asequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,99% of identity over amino acid residues 121 to 135 or from 121 to 136of human GPVI (SEQ ID NO: 13); and at least one amino acid residue fromamino acid residues 169 to 180 of human GPVI (SEQ ID NO: 13) or from asequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%,99% of identity over amino acid residues 169 to 180 of human GPVI (SEQID NO: 13).

In one embodiment, the conformational epitope comprises:

-   -   at least one (e.g., 1, 2, 3, 4, or 5) of the following residues        in GPVI sequence (SEQ ID NO: 13): 125S, 126S, 128G, 133Q, and/or        136T; and    -   at least one (e.g., 1, 2 or 3) of the following residues in GPVI        sequence (SEQ ID NO: 13):171T, 172A and/or 174H.

In one embodiment, the conformational epitope comprises:

-   -   at least one (e.g., 1, 2, 3, or 4) of the following residues in        GPVI sequence (SEQ ID NO: 13): 125S, 126S, 128G, and/or 133Q;        and    -   at least one (e.g., 1, 2 or 3) of the following residues in GPVI        sequence (SEQ ID NO: 13):171T, 172A and/or 174H.

In one embodiment, the conformational epitope comprises:

-   -   the following residues in GPVI sequence (SEQ ID NO: 13): 125S,        126S, 128G, 133Q, and 136T; and    -   the following residues in GPVI sequence (SEQ ID NO: 13):171T,        172A and 174H.

In one embodiment, the conformational epitope comprises:

-   -   the following residues in GPVI sequence (SEQ ID NO: 13): 125S,        126S, 128G, and 133Q; and    -   the following residues in GPVI sequence (SEQ ID NO: 13):171T,        172A and 174H.

In one embodiment, said conformational epitope consists of:

-   -   amino acid residues 121 to 135 or from 121 to 136 of human GPVI        (SEQ ID NO: 13) or a sequence sharing at least 60%, 70%, 75%,        80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid        residues 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:        13); and    -   amino acid residues 169 to 183 of human GPVI (SEQ ID NO: 13) or        a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%,        97%, 98%, 99% of identity over amino acid residues 169 to 183 of        human GPVI (SEQ ID NO: 13).

Thus, in one embodiment, the protein binds to a conformational epitopeconsisting of:

-   -   amino acid residues 121 to 135 or from 121 to 136 of human GPVI        (SEQ ID NO: 13) or a sequence sharing at least 60%, 70%, 75%,        80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid        residues 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:        13); and    -   amino acid residues 169 to 183 of human GPVI (SEQ ID NO: 13) or        a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%,        97%, 98%, 99% of identity over amino acid residues 169 to 183 of        human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope consists of:

-   -   amino acid residues 121 to 135 or from 121 to 136 of human GPVI        (SEQ ID NO: 13) or a sequence sharing at least 60%, 70%, 75%,        80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid        residues 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:        13); and    -   amino acid residues 169 to 180 of human GPVI (SEQ ID NO: 13) or        a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%,        97%, 98%, 99% of identity over amino acid residues 169 to 180 of        human GPVI (SEQ ID NO: 13).

Thus, in one embodiment, the protein binds to a conformational epitopeconsisting of:

-   -   amino acid residues 121 to 135 or from 121 to 136 of human GPVI        (SEQ ID NO: 13) or a sequence sharing at least 60%, 70%, 75%,        80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid        residues 121 to 135 or from 121 to 136 of human GPVI (SEQ ID NO:        13); and    -   amino acid residues 169 to 180 of human GPVI (SEQ ID NO: 13) or        a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%,        97%, 98%, 99% of identity over amino acid residues 169 to 180 of        human GPVI (SEQ ID NO: 13).

In one embodiment, the isolated protein binding to human GPVI for use inthe present invention has a K_(D) for binding to human GPVI, preferablysoluble human GPVI, less than or equal to 15 nM, preferably less than orequal to 10 nM and more preferably less than or equal to 5 nM.

In one embodiment, the isolated protein has a k_(off) for binding tohuman GPVI of less than or equal to about 8·10⁻⁴ sec⁻¹, preferably lessthan or equal to about 6·10⁻⁴ sec⁻¹, and more preferably less than orequal to about 4·10⁻⁴ sec⁻¹.

In one embodiment, the isolated protein has a k_(on) for binding tohuman GPVI of at least about 5×10⁴ M⁻¹ sec⁻¹, preferably at least about5.5×10⁴ M⁻¹ sec⁻¹, and more preferably at least about 5.9×10⁴ M⁻¹ sec⁻¹and more preferably at least about 6×10⁻⁴ sec⁻¹.

In one embodiment, the K_(D) may be determined by Surface plasmonresonance (SPR, BIAcore), using immobilized soluble GPVI at a doseranging from about 700 to about 1600 resonance units (RU) (correspondingto about 8.5 to about 11.3 fmol/mm²), preferably from about 850 to about1200 RU, more preferably from about 950 to about 1075 RU and/or usingPBS pH 7.4 as running buffer, and/or using BIAevaluation version 3.0software for analyzing data. In one embodiment, soluble GPVI correspondsto the extracellular domain of GPVI fused at its C-terminus via a linker(such as, for example, via a Gly-Gly-Arg linker) to a hFc sequence. Thissoluble GPVI may be referred to as soluble GPVI-Fc.

Methods for determining the affinity of a protein for a ligand are wellknown in the art. An example of a method for determining the affinity ofa protein for soluble GPVI is shown below:

Binding of the protein to soluble human GPVI is analyzed with surfaceplasmon resonance using a BIAcore 2000 system (Uppsala, Sweden).

Soluble GPVI-Fc is immobilized at a dose ranging from about 700 to about1600 RU (corresponding to about 8.5 to about 11.3 fmol/mm²), preferablyfrom about 850 to about 1200 RU, more preferably from about 950 to about1075 RU, and even more preferably from about 960 to about 1071 RU onto aCarboxy-Methyl Dextran CMS sensor chip using the amine coupling method(Wizard procedure). The protein is then passed over the immobilizedGPVI-Fc in PBS pH 7.4 (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.42at 25.4° C.) at a flow rate of 20 μL/min at 25° C. Kinetic constants(k_(on), k_(off)) and affinity are determined using BIAevaluationversion 3.0 software, by fitting data to binding model. PBS pH 7.4 isthe running buffer.

In an embodiment, the protein for use in the present invention is anantibody molecule or a fragment thereof selected from the groupconsisting of a whole antibody, a humanized antibody, a single chainantibody, a dimeric single chain antibody, a Fv, a Fab, a F(ab)′₂, adefucosylated antibody, a bi-specific antibody, a diabody, a triabodyand a tetrabody.

In another embodiment, the protein for use in the present invention isan antibody fragment selected from the group consisting of a unibody, adomain antibody, and a nanobody.

In another embodiment, the protein for use in the present invention isan antibody mimetic selected from the group consisting of an affibody,an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, ananticalin, an avimer, a fynomer, a versabody and a duocalin.

A domain antibody is well known in the art and refers to the smallestfunctional binding units of antibodies, corresponding to the variableregions of either the heavy or light chains of antibodies.

A nanobody is well known in the art and refers to an antibody-derivedtherapeutic protein that contains the unique structural and functionalproperties of naturally-occurring heavy chain antibodies. These heavychain antibodies may contain a single variable domain (VHH) and twoconstant domains (CH2 and CH3).

A unibody is well known in the art and refers to an antibody fragmentlacking the hinge region of IgG4 antibodies. The deletion of the hingeregion results in a molecule that is essentially half the size oftraditional IgG4 antibodies and has a univalent binding region ratherthan the bivalent biding region of IgG4 antibodies.

An affibody is well known in the art and refers to affinity proteinsbased on a 58 amino acid residue protein domain, derived from one of theIgG binding domain of staphylococcal protein A.

DARPins (Designed Ankyrin Repeat Proteins) are well known in the art andrefer to an antibody mimetic DRP (designed repeat protein) technologydeveloped to exploit the binding abilities of non-antibody proteins.

Anticalins are well known in the art and refer to another antibodymimetic technology, wherein the binding specificity is derived fromlipocalins. Anticalins may also be formatted as dual targeting protein,called Duocalins.

Avimers are well known in the art and refer to another antibody mimetictechnology.

Versabodies are well known in the art and refer to another antibodymimetic technology. They are small proteins of 3-5 kDa with >15%cysteines, which form a high disulfide density scaffold, replacing thehydrophobic core the typical proteins have.

In one embodiment, the protein for use in the present invention ismonovalent, and is preferably selected from a whole monovalent antibody,a humanized monovalent antibody, a single chain antibody, a Fv, a Fab,or an antibody fragment selected from the group consisting of a unibody,a domain antibody, and a nanobody; or a monomeric antibody mimeticselected from the group consisting of an affibody, an affilin, anaffitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, anavimer, a fynomer, and a versabody.

In another embodiment, the protein for use in the present invention isan immunoconjugate comprising an antibody or fragment thereof conjugatedto a therapeutic agent.

In another embodiment, the protein for use in the present invention is aconjugate comprising a protein conjugated to an imaging agent. Saidprotein could be used for example for imaging applications.

In an embodiment, the protein for use in the present invention is amonoclonal antibody or a fragment thereof.

In another embodiment, the protein for use in the present invention is apolyclonal antibody or a fragment thereof.

Another object of the invention is thus an anti-hGPVI antibody orantigen binding fragment thereof for treating, or for use in treating, aGPVI-related condition in a subject in need thereof, wherein saidanti-hGPVI antibody or antigen binding fragment thereof is administered(or is to be administered) during at least 2 hours to the subject,preferably during at least 4 to 6 hours.

In one embodiment, the variable region of the heavy chain of theanti-hGPVI antibody or antigen binding fragment thereof for use in thepresent invention comprises at least one of the followings CDRs:

VH-CDR1: (SEQ ID NO: 1) GYTFTSYNM; VH-CDR2: (SEQ ID NO: 2)GIYPGNGDTSYNQKFQG; and VH-CDR3: (SEQ ID NO: 3) GTVVGDWYFDV.

CDR numbering and definition are according to the Kabat/Chothiadefinition.

In one embodiment, the variable region of the light chain of theanti-hGPVI antibody or antigen binding fragment thereof for use in thepresent invention comprises at least one of the followings CDRs:

VL-CDR1: (SEQ ID NO: 4) RSSQSLENSNGNTYLN; VL-CDR2: (SEQ ID NO: 5)RVSNRFS; and VL-CDR3: (SEQ ID NO: 6) LQLTHVPWT.

CDR numbering and definition are according to the Kabat/Chothiadefinition.

In one embodiment, the anti-hGPVI antibody or antigen binding fragmentthereof for use in the present invention comprises:

-   -   in the heavy chain, at least one of the following CDR:        GYTFTSYNMH (SEQ ID NO: 1), GIYPGNGDTSYNQKFQG (SEQ ID NO: 2) and        GTVVGDWYFDV (SEQ ID NO: 3); and/or    -   in the light chain, at least one of the following CDR:        RSSQSLENSNGNTYLN (SEQ ID NO: 4), RVSNRFS (SEQ ID NO: 5), and        LQLTHVPWT (SEQ ID NO: 6).

In another embodiment of the invention, the anti-hGPVI antibody orantigen binding fragment thereof for use in the present inventioncomprises in its heavy chain the 3 CDRs SEQ ID NO: 1, SEQ ID NO: 2 andSEQ ID NO: 3.

In another embodiment of the invention, the anti-hGPVI antibody orantigen binding fragment thereof for use in the present inventioncomprises in its light chain the 3 CDRs SEQ ID NO: 4, SEQ ID NO: 5 andSEQ ID NO: 6.

In another embodiment of the invention, the anti-hGPVI antibody orantigen binding fragment thereof for use in the present inventioncomprises in its heavy chain the 3 CDRs SEQ ID NO: 1, SEQ ID NO: 2 andSEQ ID NO: 3, and in its light chain the 3 CDRs SEQ ID NO: 4, SEQ ID NO:5 and SEQ ID NO: 6.

In another embodiment of the invention, the anti-hGPVI antibody orantigen binding fragment thereof for use in the present inventioncomprises in its heavy chain the 3 CDRs SEQ ID NO: 1, SEQ ID NO: 2 andSEQ ID NO: 3, and in its light chain the 3 CDRs SEQ ID NO: 4, SEQ ID NO:5 and SEQ ID NO: 6, optionally wherein one, two, three or more of theamino acids in any of said sequences may be substituted by a differentamino acid.

According to the invention, any of the CDRs 1, 2 and 3 of the heavy andlight chains may be characterized as having an amino acid sequence thatshares at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% ofidentity with the particular CDR or sets of CDRs listed in thecorresponding SEQ ID NO.

In another embodiment of the invention, the anti-hGPVI antibody orantigen binding fragment thereof for use in the present invention is anantibody having:

-   -   (i) the heavy chain CDR 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3)        amino acid sequences as shown in SEQ ID NO: 1, 2 and 3        respectively; and    -   (ii) the light chain CDR 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3)        amino acid sequences as shown in SEQ ID NO: 4, 5 and 6        respectively;        optionally wherein one, two, three or more of the amino acids in        any of said sequences may be substituted by a different amino        acid.

In one embodiment, the anti-GPVI antibody or antigen binding fragmentthereof for use in the present invention comprises a heavy chainvariable region comprising or consisting of the sequence SEQ ID NO: 7.

(SEQ ID NO: 7) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGGIYPGNGDTSYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGT VVGDWYFDVWGQGTLVTVSS.

In one embodiment, the anti-GPVI antibody or antigen binding fragmentthereof for use in the present invention comprises a light chainvariable region comprising or consisting of the sequence SEQ ID NO: 8.

(SEQ ID NO: 8) DIQMTQSPSSLSASVGDRVTITCRSSQSLENSNGNTYLNWYQQKPGKAPKLLIYRVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQLTHVP WTFGQGTKVEITR.

In one embodiment, the anti-GPVI antibody or antigen binding fragmentthereof for use in the present invention comprises a light chainvariable region comprising or consisting of the sequence SEQ ID NO: 9.

(SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITCSASQSLENSNGNTYLNWYQQKPGKAPKLLIYRVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQLTHVP WTFGQGTKVEIKR.

In another embodiment of the invention, the anti-GPVI antibody (ACT017antibody) or antigen binding fragment thereof for use in the presentinvention comprises a heavy chain variable region comprising orconsisting of the sequence SEQ ID NO: 7 and the light chain variableregion comprising or consisting of the sequence SEQ ID NO: 8.

In another embodiment of the invention, the anti-GPVI antibody (ACT006antibody) or antigen binding fragment thereof for use in the presentinvention comprises the heavy chain variable region comprising orconsisting of the sequence SEQ ID NO: 7 and the light chain variableregion comprising or consisting of the sequence SEQ ID NO: 9.

According to the invention, one, two, three or more of the amino acidsof the heavy chain or light chain variable regions as describedhereinabove may be substituted by a different amino acid.

In another embodiment, an antibody (or a fragment thereof) for use inthe present invention comprises heavy and light chain variable regionscomprising amino acid sequences that are homologous to the amino acidsequences of the ACT017 antibody described herein, and wherein theantibodies retain the desired functional properties of the proteindescribed in the present invention.

In another embodiment, an antibody (or a fragment thereof) for use inthe present invention comprises heavy and light chain variable regionscomprising amino acid sequences that are homologous to the amino acidsequences of the ACT006 antibody described herein, and wherein theantibodies retain the desired functional properties of the proteindescribed in the present invention.

According to the invention, the heavy chain variable region encompassessequences that have 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% ormore of identity with SEQ ID NO: 7.

According to the invention, the light chain variable region encompassessequences that have 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% ormore of identity with SEQ ID NO: 8 or SEQ ID NO: 9.

In any of the antibodies for use in the present invention (e.g. ACT017or ACT006), the specified variable region and CDR sequences may compriseconservative sequence modifications. Conservative sequence modificationsrefer to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody for use in the present invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are typically thosein which an amino acid residue is replaced with an amino acid residuehaving a side chain with similar physicochemical properties. Specifiedvariable region and CDR sequences may comprise one, two, three, four ormore amino acid insertions, deletions or substitutions. Wheresubstitutions are made, preferred substitutions will be conservativemodifications. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody foruse in the present invention can be replaced with other amino acidresidues from the same side chain family and the altered antibody can betested for retained function (i.e., the properties set forth herein)using the assays described herein.

In one embodiment, the antibody (or fragment thereof) for use in thepresent invention binds essentially the same epitope as ACT017 or ACT006antibodies. In the present invention, an antibody that binds essentiallythe same epitope as ACT017 or ACT006 antibodies will be referred as anACT017-like or ACT006-like antibody, respectively.

In one embodiment, the antibody (or fragment thereof) for use in thepresent invention competes for binding to hGPVI with an antibody asdescribed hereinabove, in particular with an antibody selected fromACT017 and ACT006.

In some embodiments of this invention, anti-hGPVI antibodies or fragmentthereof comprising VH and VL domains, or CDRs thereof may comprise CH1domains and/or CL domains, the amino acid sequence of which is fully orsubstantially human. Where the GPVI binding protein is an antibodyintended for human therapeutic use, it is typical for the entireconstant region of the antibody, or at least a part thereof, to have afully or substantially human amino acid sequence. Therefore, one or moreor any combination of the CH1 domain, hinge region, CH2 domain, CH3domain and CL domain (and CH4 domain if present) may be fully orsubstantially human with respect to its amino acid sequence.Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may all have a fully orsubstantially human amino acid sequence. In the context of the constantregion of a humanized or chimeric antibody, or an antibody fragment, theterm “substantially human” refers to an amino acid sequence identity ofat least 70%, or at least 80%, or at least 90%, or at least 95%, or atleast 97%, or at least 99% with a human constant region. The term “humanamino acid sequence” in this context refers to an amino acid sequencewhich is encoded by a human immunoglobulin gene, which includesgermline, rearranged and somatically mutated genes. The invention alsocontemplates the use of proteins comprising constant domains of “human”sequence which have been altered, by one or more amino acid additions,deletions or substitutions with respect to the human sequence, exceptingthose embodiments where the presence of a “fully human” hinge region isexpressly required. The presence of a “fully human” hinge region in theanti-hGPVI antibodies for use in the present invention may be beneficialboth to minimize immunogenicity and to optimize stability of theantibody. It is considered that one or more amino acid substitutions,insertions or deletions may be made within the constant region of theheavy and/or the light chain, particularly within the Fc region. Aminoacid substitutions may result in replacement of the substituted aminoacid with a different naturally occurring amino acid, or with anon-natural or modified amino acid. Other structural modifications arealso permitted, such as for example changes in glycosylation pattern(e.g., by addition or deletion of N- or O-linked glycosylation sites).Depending on the intended use of the antibody, it may be desirable tomodify the antibody with respect to its binding properties to Fcreceptors, for example to modulate effector function. For examplecysteine residue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved effector function. See Caronet al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992).

In one embodiment, the heavy chain variable region of sequence SEQ IDNO: 7 is encoded by SEQ ID NO: 10:

(SEQ ID NO: 10) CAGGTTCAGCTGGTTCAGTCAGGGGCTGAGGTGAAGAAGCCTGGAGCCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAGACAGGCTCCTGGACAGGGCCTGGAATGGATGGGAGGTATTTATCCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCCAGGGCCGAGTTACTATGACTCGGGACACTTCCACCTCTACAGTGTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACCGCGGTCTATTACTGTGCAAGAGGCACCGTGGTCGGCGACTGGTACTTCGATGTGTGGGGCCAAGGCACCCTGGTCAC CGTGAGCAGT.

In one embodiment, the light chain variable region of sequence SEQ IDNO: 8 is encoded by SEQ ID NO: 11:

(SEQ ID NO: 11) GACATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGAAGTAGTCAGAGCCTTGAGAACAGCAACGGAAACACCTACCTGAATTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCTGCTGATCTACAGAGTTTCCAACCGATTCTCTGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTTCACCTTCACCATCAGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCTCCAGCTGACTCATGTCCCATGGACCTTCGGTCAGGGCACCAAGGTGGAGATCACCCGG.

In one embodiment, the light chain variable region of sequence SEQ IDNO: 9 is encoded by SEQ ID NO: 12.

(SEQ ID NO: 12) GACATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGTGCCAGTCAGAGCCTTGAGAACAGCAACGGAAACACCTACCTGAATTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCTGCTGATCTACAGAGTTTCCAACCGATTCTCTGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTTCACCCTCACCATCAGCAGCCTCCAGCCAGAGGACTTCGCCACCTACTACTGCCTCCAGCTGACTCATGTCCCATGGACCTTCGGTCAGGGCACCAAGGTGGAGATCAAACGC.

In one embodiment, the nucleic sequences encoding the anti-GPVI antibodyor fragment thereof for use in the present invention are comprised in anexpression vector. In one embodiment, the expression vector comprises atleast one of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 or anysequence having a nucleic acid sequence that shares at least 60%, 70%,75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity with said SEQ ID NO:10-12.

In one embodiment, the vector comprises the sequence SEQ ID NO: 10 and asequence encoding a constant region of a heavy chain. A non-limitingexample of a sequence encoding a constant region of a heavy chain is SEQID NO: 14.

(SEQ ID NO: 14) GCCTCCACCAAGGGTCCCTCAGTCTTCCCACTGGCACCCTCCTCCAAGAGCACCTCTGGTGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCAGTGACTGTGTCATGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCTGCTGTCTTGCAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTCGAGCCTAAGTCATGCGACAAGACTCAC.

In one embodiment, the vector comprises the sequence SEQ ID NO: 11 orSEQ ID NO: 12 and a sequence encoding a constant region of a lightchain. A non-limiting example of a sequence encoding a constant regionof a light chain is SEQ ID NO: 15.

(SEQ ID NO: 15) ACTGTGGCTGCACCAAGTGTGTTCATCTTCCCACCTAGCGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTCCTGAACAACTTCTACCCACGGGAGGCCAAGGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAAGATAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACTCTGAGCAAAGCAGACTACGAGAAGCACAAGGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCCCCTGTCACAAAGAGC TTCAACCGGGGAGAGTGT.

In one embodiment, the vector comprises a sequence encoding a signalpeptide. Non-limiting examples of signal peptides sequences include, butare not limited to, SEQ ID NO: 16 and SEQ ID NO: 17.

(SEQ ID NO: 16) ATGGATATGCGTGTACCAGCTCAACTACTTGGACTTCTATTGCTTTGGCTTCGTGGTGCTAGATGT. (SEQ ID NO: 17)ATGGACTGGACTTGGAGAATCCTATTCTTGGTTGCTGCAGCTACAGGTGC TCATTCA.

In one embodiment, the vector comprises SEQ ID NO: 10, and a sequenceencoding a constant region of a heavy chain (such as, for example, SEQID NO: 14), and a signal peptide sequence. An example of such a vectoris a vector comprising SEQ ID NO: 18. SEQ ID NO: 18 further comprisescloning sites.

(SEQ ID NO: 18) GCGGCCGCCACCATGGACTGGACTTGGAGAATCCTATTCTTGGTTGCTGCAGCTACAGGTGCTCATTCACAGGTTCAGCTGGTTCAGTCAGGGGCTGAGGTGAAGAAGCCTGGAGCCTCAGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAGACAGGCTCCTGGACAGGGCCTGGAATGGATGGGAGGTATTTATCCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCCAGGGCCGAGTTACTATGACTCGGGACACTTCCACCTCTACAGTGTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACCGCGGTCTATTACTGTGCAAGAGGCACCGTGGTCGGCGACTGGTACTTCGATGTGTGGGGCCAAGGCACCCTGGTCACCGTGAGCAGTGCCTCCACCAAGGGTCCCTCAGTCTTCCCACTGGCACCCTCCTCCAAGAGCACCTCTGGTGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCAGTGACTGTGTCATGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCTGCTGTCTTGCAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTCGAGCCTAAGTCATGCGACAAGACTCAC TGATGAGGATCC.

In one embodiment, the vector comprises SEQ ID NO: 8, and a sequenceencoding a constant region of a light chain (such as, for example, SEQID NO: 15), and a signal peptide sequence. An example of such a vectoris a vector comprising SEQ ID NO: 19. SEQ ID NO: 19 further comprisescloning sites.

(SEQ ID NO: 19) GACGTCACCATGGATATGCGTGTACCAGCTCAACTACTTGGACTTCTATTGCTTTGGCTTCGTGGTGCTAGATGTGACATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGAAGTAGTCAGAGCCTTGAGAACAGCAACGGAAACACCTACCTGAATTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCTGCTGATCTACAGAGTTTCCAACCGATTCTCTGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTTCACCTTCACCATCAGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCTCCAGCTGACTCATGTCCCATGGACCTTCGGTCAGGGCACCAAGGTGGAGATCACCCGGACTGTGGCTGCACCAAGTGTGTTCATCTTCCCACCTAGCGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTCCTGAACAACTTCTACCCACGGGAGGCCAAGGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAAGATAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACTCTGAGCAAAGCAGACTACGAGAAGCACAAGGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCCCCTGTCACAAAGAGCTTCAACCGGGGAGAGTGTTGATGATATC.

In one embodiment, the vector comprises SEQ ID NO: 9, and a sequenceencoding a constant region of a light chain (such as, for example, SEQID NO: 15), and a signal peptide sequence. An example of such a vectoris a vector comprising SEQ ID NO: 20.

SEQ ID NO: 20 further comprises cloning sites.

(SEQ ID NO: 20) GACGTCACCATGGATATGCGTGTACCAGCTCAACTACTTGGACTTCTATTGCTTTGGCTTCGTGGTGCTAGATGTGACATCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGTGCCAGTCAGAGCCTTGAGAACAGCAACGGAAACACCTACCTGAATTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCTGCTGATCTACAGAGTTTCCAACCGATTCTCTGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTTCACCCTCACCATCAGCAGCCTCCAGCCAGAGGACTTCGCCACCTACTACTGCCTCCAGCTGACTCATGTCCCATGGACCTTCGGTCAGGGCACCAAGGTGGAGATCAAACGCACTGTGGCTGCACCAAGTGTGTTCATCTTCCCACCTAGCGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTCCTGAACAACTTCTACCCACGGGAGGCCAAGGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAAGATAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACTCTGAGCAAAGCAGACTACGAGAAGCACAAGGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCCCCTGTCACAAAGAGCTTCAACCGGGGAGAGTGTTGATGATATC.

In one embodiment, the vector as described hereinabove is comprised inan isolated host cell. Said host cell may be used for the recombinantproduction of the antibodies for use in the present invention. In anembodiment, host cells may be prokaryote, yeast, or eukaryote cellspreferably mammalian cells, such as, for example: monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc.Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather,Biol. Reprod. 23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCCCRL 1581; ATCC CRL 8287) or NSO (HPA culture collections no. 85110503);monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), aswell as DSM's PERC-6 cell line. Expression vectors suitable for use ineach of these host cells are also generally known in the art. It shouldbe noted that the term “host cell” generally refers to a cultured cellline. Whole human beings into which an expression vector encoding anantigen binding protein for use according to the invention has beenintroduced are explicitly excluded from the definition of a “host cell”.

Methods for producing an anti-hGPVI antibody or antigen binding fragmentthereof for use in the present invention are well known in the art.Examples of suitable methods comprise culturing host cells containingthe isolated polynucleotide sequence encoding the anti-hGPVI antibody orfragment thereof for use in the present invention under conditionssuitable for expression of the anti-hGPVI antibody or fragment thereof,and recovering the expressed anti-hGPVI antibody or fragment thereof.This recombinant process can be used for large scale production ofanti-hGPVI antibodies or fragment thereof for use in the presentinvention, including monoclonal antibodies intended for in vivotherapeutic uses. These processes are available in the art and will beknown by the skilled person.

In one embodiment, the protein for use in the present invention may bepurified by chromatography, preferably by affinity chromatography, morepreferably by affinity chromatography on protein L agarose.

Therefore, in one embodiment, the protein for use in the presentinvention comprises a domain for binding protein L (PpL). Methods fortransferring PpL-binding activity onto proteins of the invention aredescribed in Muzard et al., Analytical Biochemistry 388, 331-338, 2009and in Lakhrif et al., MAbs. 2016; 8(2):379-88, which are incorporatedherein by reference.

Fragments and derivatives of antibodies for use in the present invention(which are encompassed by the term “antibody” or “antibodies” as used inthis application, unless otherwise stated or clearly contradicted bycontext), preferably a ACT017-like or ACT006-like antibody, can beproduced by techniques that are known in the art. “Fragments” comprise aportion of the intact antibody, generally the antigen binding site orvariable region. Examples of antibody fragments include Fab, Fab′,Fab′-SH, F (ab′)₂, and Fv fragments; diabodies; any antibody fragmentthat is a protein having a primary structure consisting of oneuninterrupted sequence of contiguous amino acid residues (referred toherein as a “single-chain antibody fragment” or “single chain protein”),including without limitation (1) single-chain Fv molecules (2) singlechain proteins containing only one light chain variable domain, or afragment thereof that contains the three CDRs of the light chainvariable domain, without an associated heavy chain moiety and (3) singlechain proteins containing only one heavy chain variable region, or afragment thereof containing the three CDRs of the heavy chain variableregion, without an associated light chain moiety; and multispecificantibodies formed from antibody fragments. Fragments of the presentantibodies can be obtained using standard methods. For instance, Fab orF(ab′)₂ fragments may be produced by protease digestion of the isolatedantibodies, according to conventional techniques. It will be appreciatedthat immunoreactive fragments can be modified using known methods, forexample to slow clearance in vivo and obtain a more desirablepharmacokinetic profile the fragment may be modified with polyethyleneglycol (PEG). Methods for coupling and site-specifically conjugating PEGto a Fab′ fragment are described in, for example, Leong et al.,Cytokines 16 (3): 106-119 (2001) and Delgado et al., Br. J. Cancer 73(2): 175-182 (1996), the disclosures of which are incorporated herein byreference.

Alternatively, the DNA encoding an antibody for use in the presentinvention, preferably an ACT017-like or ACT006-like antibody, may bemodified so as to encode a fragment. The modified DNA is then insertedinto an expression vector and used to transform or transfect anappropriate cell, which then expresses the desired fragment.

Another object of the invention is a composition for treating or for usein treating a GPVI-related condition in a subject in need thereof,wherein said composition comprises at least one isolated humanizedprotein binding to human Glycoprotein VI (hGPVI) as described hereabove, and wherein said composition is administered (or is to beadministered) during at least 2 hours to the subject, preferably duringat least 4 to 6 hours.

Another object of the invention is a pharmaceutical composition fortreating or for use in treating a GPVI-related condition in a subject inneed thereof, wherein said pharmaceutical composition comprises at leastone isolated humanized protein binding to human Glycoprotein VI (hGPVI)as described here above and at least one pharmaceutically acceptableexcipient, and wherein said pharmaceutical composition is administered(or is to be administered) during at least 2 hours to the subject,preferably during at least 4 to 6 hours.

Pharmaceutically acceptable excipients that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as acetates, citrates, phosphates,glycine, sorbic acid, potassium sorbate, sugars such as glucose, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances (for example sodium carboxymethylcellulose), polyethyleneglycol, polyacrylates, waxes, polyethylene-polyoxypropylene-blockpolymers, polyethylene glycol and wool fat.

Another object of the invention is a pharmaceutical composition fortreating or for use in treating a GPVI-related condition in a subject inneed thereof, wherein the pH of the pharmaceutical composition iscomprised between 4 to 6 and preferably is of about 5.0.

Another object of the invention is a medicament for treating or for usein treating a GPVI-related condition in a subject in need thereof,wherein said medicament comprises at least one isolated humanizedprotein binding to human Glycoprotein VI (hGPVI) as described hereabove, and wherein said medicament is administered (or is to beadministered) during at least 2 hours to the subject, preferably duringat least 4 to 6 hours.

In one embodiment, the composition, pharmaceutical composition ormedicament for use in the present invention further comprises anotheragent useful for treating a GPVI-related condition, such as, for examplea thrombolytic agent. One example of thrombolytic agent is t-PA,including native t-PA and recombinant t-PA, as well as modified forms oft-PA that retain the enzymatic and/or fibrinolytic activity of nativet-PA. As used herein, t-PA has its general meaning in the art and refersto tissue-type plasminogen activator. The enzymatic activity of amodified form of t-PA may be measured by assessing the ability of themolecule to convert plasminogen to plasmin. The fibrinolytic activity ofa modified form of t-PA may be determined by any in vitro clot lysistest known in the art. T-PA is commercially available as alteplase(Activase® or Actilyse®). Recombinant t-PA has been described in the artand is known by the skilled artisan. Modified forms of t-PA has beendescribed in the art and is known by the skilled artisan, and include,without limitation, t-PA having deleted or substituted amino acids ordomains, modified glycosylation status, and variants conjugated to orfused with other molecules. Examples of modified forms of t-PA weredescribed, for example, in EP0352119, WO93/24635, EP0382174 andWO2013/034710, which are incorporated herein by reference.

In one embodiment, the composition, pharmaceutical composition ormedicament for use in the present invention further comprises a mutatedt-PA as described in WO2013/034710.

In one embodiment, said mutated t-PA comprises the sequence SEQ ID NO:25 (corresponding to human wt t-PA mature form) or SEQ ID NO: 26(corresponding to human wt t-PA first chain of tc-t-PA), preferablyconsisting of SEQ ID NO: 25 or of the association of SEQ ID NO:26 andSEQ ID NO:27 (corresponding to human wt t-PA second chain of tc-t-PA),or variant thereof having at least 80% identity, wherein said sequencecomprises a mutation consisting of the replacement of any amino acid ofthe Lysine Binding Site of SEQ ID NO: 25 or SEQ ID NO:26 by ahydrophilic amino acid chosen from arginine, aspartic acid, glutamicacid, lysine, asparagine, glutamine, serine, threonine, tyrosine andhistidine, preferably by arginine, and/or a mutation consisting of thereplacement of arginine in position 275 of SEQ ID NO: 25 or SEQ ID NO:26by serine.

In one embodiment, said mutated t-PA comprises the sequence SEQ ID NO:25 (corresponding to human wt t-PA mature form) or SEQ ID NO: 26(corresponding to human wt t-PA first chain of two chain t-PA),preferably consisting of SEQ ID NO: 25 or of the association of SEQ IDNO:26 and SEQ ID NO:27 (corresponding to human wt t-PA second chain oftwo chain t-PA), or variant thereof having at least 80% identity,wherein said sequence comprises a mutation consisting of the replacementof tryptophan in position 253 of SEQ ID NO: 25 or SEQ ID NO:26 by ahydrophilic amino acid chosen from arginine, aspartic acid, glutamicacid, lysine, asparagine, glutamine, serine, threonine, tyrosine andhistidine, preferably by arginine, and/or a mutation consisting of thereplacement of arginine in position 275 of SEQ ID NO: 25 or SEQ ID NO:26by serine.

In one embodiment, said mutated t-PA further comprises at least one ofthe following mutations:

-   -   the replacement of proline in position 125 of SEQ ID NO:25 or        SEQ ID NO:26 by arginine,    -   the deletion of the Finger domain in the N-terminus and/or the        deletion of the EGF-like domain, in SEQ ID NO:25 or SEQ ID        NO:26, and/or the replacement of asparagine in position 117 of        SEQ ID NO:25 or SEQ ID NO:26 by glutamine,    -   the replacement of threonine in position 103 of SEQ ID NO:25 or        SEQ ID NO:26 by asparagine, and/or the replacement of asparagine        in position 117 of SEQ ID NO:25 or SEQ ID NO:26 by glutamine,        and/or the replacement of lysine-histidine-arginine-arginine        (KHRR) in positions 296 to 299 of SEQ ID NO:25 by        alanine-alanine-alanine-alanine (AAAA),    -   the replacement of cysteine in position 84 of SEQ ID NO:25 or        SEQ ID NO:26 by serine,    -   the replacement of arginine in position 275 of SEQ ID NO:25 or        SEQ ID NO:26 by glutamic acid or glycine, and/or the deletion of        the Kringle 1 domain in SEQ ID NO:25 or SEQ ID NO:26.

Therefore, in one embodiment, the composition, pharmaceuticalcomposition or medicament for use in the present invention comprises atleast one isolated humanized protein binding to human Glycoprotein VI(hGPVI) as described here above, preferably at least one anti-hGPVIantibody as described hereinabove or antigen binding fragment thereof,and t-PA (either in a native, recombinant or modified form).

One object of the invention is a kit of part comprising, in a firstpart, at least one isolated humanized protein binding to humanGlycoprotein VI (hGPVI) as described hereinabove (preferably at leastone isolated anti-hGPVI antibody or antigen binding fragment thereof asdescribed hereinabove) and, in a second part, at least one t-PA (eitherin a native, recombinant or modified form).

Another object is thus a kit of part as described hereinabove, for usein the treatment of a GPVI-related condition in a subject in needthereof.

In an embodiment, the protein for use in the present invention is ananti-hGPVI antibody or antigen binding fragment thereof that inhibitsGPVI signaling and/or signaling of a ligand of GPVI. As used herein, theterm “inhibit” means that the protein for use in the present inventionis capable of blocking, reducing, preventing or neutralizing GPVIinteraction with its ligands, GPVI signaling and/or the activation ofmolecules from the GPVI signaling pathway.

Examples of ligands of GPVI include, but are not limited to, collagen,fibrin, fibronectin, vitronectin and laminins.

In an embodiment, the protein for use in the present invention is aneutralizing anti-hGPVI antibody or fragment thereof.

In an embodiment, the protein for use in the present invention inhibitsthe binding of GPVI to a ligand thereof (such as, for example, collagen,fibrin or any other GPVI ligand capable of inducing downstream signalingand platelet activation).

In an embodiment, the protein for use in the present invention inhibitsand/or prevents platelet activation, secretion and aggregation inresponse to a ligand of GPVI, such as, for example, collagen. In anembodiment, the protein for use in the present invention inhibits and/orprevents platelet adhesion to a ligand of GPVI, such as, for example,collagen.

In an embodiment, the protein for use in the present invention inhibitsand/or prevents GPVI-dependent thrombin production in response to aligand of GPVI, such as, for example, fibrin. In an embodiment, theprotein for use in the present invention inhibits and/or preventplatelet-catalyzed thrombin production in response to collagen and/or totissue factor.

In an embodiment, the protein for use in the present invention inhibitsand/or prevents platelet recruitment by a ligand of GPVI (such as, forexample, fibrin) via GPVI.

In one embodiment, the protein for use in the present invention inducessaturation of platelets in whole blood or in platelet rich plasma whenpresent at a concentration ranging from about 1 to about 200 μg/mL,preferably from about 2 to about 100 μg/mL, and more preferably fromabout 5 to about 50 μg/mL.

In one embodiment, the protein for use in the present invention inhibitscollagen-induced platelet aggregation when used at a concentration of atleast about 15 μg/mL, preferably of at least about 10 μg/mL. Preferably,the protein for use in the present invention fully inhibitscollagen-induced platelet aggregation when used at such concentrations.

In one embodiment, the IC50 of the protein for use in the presentinvention for inhibiting collagen-induced platelet aggregation rangesfrom about 0.5 to about 10 μg/mL, preferably from about 1 to about 6μg/mL, more preferably from about 2 to about 3.2 μg/mL.

In one embodiment, the concentration of the protein for use in thepresent invention reducing by 50% the velocity of collagen-inducedplatelet aggregation ranges from about 0.5 to about 5 μg/mL, preferablyfrom about 1 to about 3 μg/mL, more preferably of about 2 μg/mL.

In one embodiment, the concentration of the protein for use in thepresent invention reducing the intensity of collagen-induced plateletaggregation ranges from about 0.5 to about 10 μg/mL, preferably fromabout 1 to about 6 μg/mL, more preferably of about 3.2 μg/mL.

In one embodiment, the protein for use in the present invention does notinduce depletion of GPVI when administered in vivo, such as, forexample, when administered at a dose ranging from 0.01 to 500 mg.

In one embodiment, the protein for use in the present invention does notinduce a decrease in platelet count, i.e., thrombocytopenia, whenadministered in vivo, such as, for example, when administered at a doseranging from 0.01 to 500 mg.

Examples of GPVI-related conditions are well known in the art andinclude, without limitation, inflammation, thrombosis, disordersassociated with abnormal or aberrant megakaryocyte and/or plateletproliferation, differentiation, morphology, migration, aggregation,degranulation and/or function, thrombotic disorders (such as, forexample, thrombotic occlusion of coronary arteries), diseases exhibitingquantitative or qualitative platelet dysfunction and diseases displayingendothelial dysfunction, cerebral vascular diseases, coronary diseases,disorders resulting from any blood vessel insult that can result inplatelet aggregation, disorders associated with aberrant signaltransduction in response to ligands of GPVI, disorders associated withaberrant levels of GPVI expression and/or activity either in cells thatnormally express GPVI or in cells that do not express GPVI, bone marrowand peripheral blood or disorders in which platelets contribute bymodulating inflammatory responses.

In one embodiment, said GPVI-related condition is a cardiovasculardisease selected from arterial and venous thrombosis, restenosis, acutecoronary syndrome, cerebrovascular accidents due to atherosclerosis,myocardial infarction, pulmonary embolism, critical limb ischemia andperipheral artery disease.

In one embodiment, said GPVI-related condition is a venous thrombosis.In one embodiment, said GPVI-related condition is a restenosis. In oneembodiment, said GPVI-related condition is an acute coronary syndrome.In one embodiment, said GPVI-related condition is cerebrovascularaccidents due to atherosclerosis. In one embodiment, said GPVI-relatedcondition is a myocardial infarction. In one embodiment, saidGPVI-related condition is a pulmonary embolism. In one embodiment, saidGPVI-related condition is a critical limb ischemia. In one embodiment,said GPVI-related condition is a peripheral artery disease.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to modulateleukocyte-platelet in inflammation and/or thrombosis. Therefore,according to an embodiment, the protein, composition, pharmaceuticalcomposition or medicament as described hereinabove is used to treatinflammation and/or thrombosis.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to modulate, preferablyto prevent, platelet adhesion aggregation and degranulation.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to treat thromboticdisorders (such as, for example, thrombotic occlusion of coronaryarteries), diseases exhibiting quantitative or qualitative plateletdysfunction and diseases displaying endothelial dysfunction. Thesediseases include, but are not limited to, coronary artery and cerebralartery diseases.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to treat cerebralvascular diseases, including ischemic stroke, venous thromboembolismdiseases (such as, for example, diseases involving leg swelling, painand ulceration, pulmonary embolism, abdominal venous thrombosis),thrombotic microangiopathies, vascular purpura.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to treat coronarydiseases (such as, for example, cardiovascular diseases includingunstable angina pectoris, myocardial infarction, acute myocardialinfarction, coronary artery disease, coronary revascularization,coronary restenosis, ventricular thromboembolism, atherosclerosis,coronary artery disease (e. g., arterial occlusive disorders), plaqueformation, cardiac ischemia, including complications related to coronaryprocedures, such as percutaneous coronary artery angioplasty (balloonangioplasty) procedures). With respect to coronary procedures, suchtreatment can be achieved via administration of a protein as describedabove prior to, during, or subsequent to the procedure. In a preferredembodiment, such administration can be utilized to prevent acute cardiacischemia following angioplasty.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to treat disordersresulting from any blood vessel insult that can result in plateletaggregation. Such blood vessel insults include, but are not limited to,vessel wall injury, such as vessel injuries that result in a highlythrombogenic surface exposed within an otherwise intact blood vessel e.g., vessel wall injuries that result in release of ADP, thrombin and/orepinephrine, fluid shear stress that occurs at the site of vesselnarrowing, ruptures and/or tears at the sites of atheroscleroticplaques, and injury resulting from balloon angioplasty or atherectomy.

Further, in certain embodiments, it is preferred that the protein doesnot affect other platelet attributes or functions, such asagonist-induced platelet shape change (e.g., GPIb-vWF-mediated plateletactivation), release of internal platelet granule components, activationof signal transduction pathways or induction of calcium mobilizationupon platelet activation by agonists that do not interact with GPVI.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to treat disordersassociated with aberrant signal transduction in response to ligands ofGPVI (including, without limitation, collagen, fibrin, fibronectin,vitronectin and laminins) or to other extracellular matrix proteins.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to treat disordersassociated with aberrant levels of GPVI expression and/or activityeither in cells that normally express GPVI or in cells that do notexpress GPVI. For example, the protein can be used to modulate disordersassociated with aberrant expression of GPVI in cancerous (e. g., tumor)cells that do not normally express GPVI. Such disorders can include, forexample, ones associated with tumor cell migration and progression tometastasis.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to modulateimmunoregulatory functions of platelets.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to treat disorders bonemarrow and peripheral blood.

In one embodiment, the protein, composition, pharmaceutical compositionor medicament as described hereinabove is used to treat disorders inwhich platelets contribute by modulating inflammatory responsesincluding, without limitation, sustained or prolonged inflammationassociated with infection, arthritis, fibrosis or disorders in whichplatelets modulate cell functions including, without limitation, cancercells proliferation and/or dissemination.

Further examples of diseases, disorders or conditions related to GPVIinclude, but are not limited to, cardiovascular diseases and/orcardiovascular events, such as, for example, arterial thrombosisincluding atherothrombosis, ischemic events, acute coronary arterysyndrome, myocardial infarction (heart attack), acute cerebrovascularischemia (stroke), percutaneous coronary intervention, stentingthrombosis, ischemic, restenosis, ischemia, (acute and chronic),diseases of the aorta and its branches (such as aortic aneurysm,thrombosis), peripheral artery disease, venous thrombosis, acutephlebitis and pulmonary embolism, cancer-associated thrombosis(Trousseau syndrome), inflammatory thrombosis and thrombosis associatedto infection.

In one embodiment, the pharmaceutical composition or medicament of theinvention is for treating or for use in treating arterial or venousthrombosis, restenosis, acute coronary syndrome or cerebrovascularaccidents due to atherosclerosis, preferably thrombosis.

In one embodiment, the subject is affected by, preferably is diagnosedwith a disease, disorder or condition related to GPVI, preferably acardiovascular disease and/or event.

In one embodiment, the subject experienced a cardiovascular event (suchas, for example, thrombosis, stroke, myocardial infarction or acerebrovascular accident) less than 48 hours, preferably less than 24hours, 12 hours or less, before the administration of the protein foruse in the present invention.

In one embodiment, the subject receive a protein, composition,pharmaceutical composition or medicament as described hereinabove aspart of a treatment protocol.

In one embodiment, said treatment protocol further comprises, before,concomitantly or after the administration of the protein of theinvention, the administration of another agent useful for treating aGPVI-related condition, such as, for example a thrombolytic agent,preferably t-PA (including native t-PA and recombinant t-PA, as well asmodified forms of t-PA that retain the enzymatic and/or fibrinolyticactivity of native t-PA).

In one embodiment, said treatment protocol further comprises, before,concomitantly or after the administration of the protein of theinvention, the treatment of the subject by endovascular treatment and/orby thrombectomy (also known as embolectomy).

Therefore, in one embodiment, the subject to be treated was previouslytreated or is to be treated by endovascular treatment and/or bythrombectomy.

In one embodiment, a dose of the protein for use in the presentinvention ranging from about 0.5 mg/kg to about 50 mg/kg is administered(or is to be administered) to the patient, preferably ranging from about1 mg/kg to about 32 mg/kg, more preferably of about 8 mg/kg. In anotherembodiment, a dose of humanized protein ranging from about 2.5 mg/kg toabout 25 mg/kg, preferably from about 5 mg/kg to about 15 mg/kg, morepreferably of about 8 mg/kg is to be administered to the patient. In oneembodiment, a dose of the protein for use in the present invention ofabout 0.5 mg/kg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or of about50 mg/kg is administered (or is to be administered) to the subject.

In one embodiment, a dose of the protein for use in the presentinvention ranging from about 30 mg to about 3000 mg is administered (oris to be administered) to the patient, preferably ranging from about 60mg to about 2000 mg, more preferably of about 100 to about 1000 mg, andeven more preferably of about 500 mg. In one embodiment, a dose of theprotein for use in the present invention of about 30, 60, 62.5, 90, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500 orof about 3000 mg is administered (or is to be administered) to thesubject. In another embodiment, a dose of the protein for use in thepresent invention ranges from about 100 mg to about 2000 mg, from about125 mg to about 2000 mg, preferably from about 250 mg to about 1000 mgor from about 500 mg to about 1000 mg.

The composition will be formulated for administration to the subject.The compositions may be administered parenterally, by inhalation spray,rectally, nasally, or via an implanted reservoir. The termadministration used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques.

In one embodiment, the protein for use in the present invention isinjected, preferably by intravenous infusion. In another embodiment, theprotein for use in the present invention is injected intraperitoneally.In another embodiment, the protein for use in the present invention isinjected intradermally.

Examples of forms adapted for injection include, but are not limited to,solutions, such as, for example, sterile aqueous solutions, gels,dispersions, emulsions, suspensions, solid forms suitable for using toprepare solutions or suspensions upon the addition of a liquid prior touse, such as, for example, powder, liposomal forms and the like.

Sterile injectable forms of the compositions may be aqueous or anoleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution and isotonicsodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

In one embodiment, the protein for use in the present invention isadministered (or is to be administered) to the subject during about 2hours, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, 11, 11.5 or during about 12 hours.

In one embodiment, the protein for use in the present invention iscontinuously administered to the subject during at least 2 hours,preferably during at least 4 to 6 hours (e.g. during about 2 hours, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5 or during about 12 hours). As used herein, the terms “continuouslyadministered” refers to the administration of a compound for a prolongedperiod of time with a substantially constant speed of administration.

In another embodiment, a first bolus of the protein is injected,followed by the continuous administration of the remaining dose of theprotein.

In one embodiment, said first bolus administration comprises about 10 to50%, preferably about 20% of the total dosage of the isolated humanizedprotein to be administered. In one embodiment, said first bolusadministration comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50% of the totaldosage of the isolated humanized protein to be administered.

In one embodiment, said first bolus is administered in about 5 to 30minutes, preferably in about 15 minutes.

In one embodiment, about 20% of the total dosage of the protein for usein the present administration are administered during the first 15minutes of the administration, followed by a continuous administrationof the remaining 80% during the next 5 hours 45 minutes.

In one embodiment, said specific administration regimen (continuedadministration during at least 2 hours, with optionally a first bolus)allows a prolonged effect of the protein, which may be observed afterthe end of administration of said protein, as demonstrated in theExamples. In one embodiment, the effect of the protein on plateletaggregation is observed for at least about 1, 2, 3, 4, 5, 6, 12, 18, 24,36 or 48 hours after the end of the administration of the protein.

In one embodiment, a protein for use in the present invention present ina pharmaceutical composition can be supplied at a concentration rangingfrom about 1 to about 100 mg/mL, such as, for example, at aconcentration of 1 mg/mL, 5 mg/mL, 10 mg/mL, 50 mg/mL or 100 mg/mL. Inone embodiment, the protein is supplied at a concentration of about 10mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.

In one embodiment, the pharmaceutical composition may comprise a proteinof the invention in PBS pH 7.2-7.7.

In one embodiment, the pharmaceutical composition may comprise a proteinof the invention in sodium citrate buffer 20 mM, NaCl 130 mM, pH 5.0.

Another object of the invention is a method of treating a GPVI-relatedcondition, wherein said method comprises administering to a subject inneed thereof an isolated humanized protein binding to hGPVI as describedhereinabove, or a composition, pharmaceutical composition or medicamentas described hereinabove. According to the invention, the method of theinvention comprises administering said protein, composition,pharmaceutical composition or medicament during at least 2 hours to thesubject, preferably during at least 4 to 6 hours.

In one embodiment, the method of treating a GPVI-related condition ofthe present invention further comprises treating the patient withendovascular treatment and/or thrombectomy. In one embodiment, saidendovascular treatment and/or thrombectomy is carried out before,concomitantly or after the administration of the protein (preferably theantibody) of the present invention.

In one embodiment, the method of treating a GPVI-related condition ofthe present invention further comprises a step of administering anotheragent useful for treating a GPVI-related condition, such as, for examplea thrombolytic agent, preferably t-PA (including native t-PA andrecombinant t-PA, as well as modified forms of t-PA that retain theenzymatic and/or fibrinolytic activity of native t-PA). In oneembodiment, said additional agent is administered before, concomitantlyor after the administration of the protein (preferably the antibody) ofthe present invention.

In one embodiment, the method of the invention comprises administering adose of the protein as described in the present invention ranging fromabout 0.5 mg/kg to about 50 mg/kg, preferably ranging from about 1 mg/kgto about 32 mg/kg, more preferably of about 8 mg/kg. In anotherembodiment, the method of the invention comprises administering a doseof the protein as described in the present invention ranging from about2.5 mg/kg to about 25 mg/kg, preferably from about 5 mg/kg to about 15mg/kg, more preferably of about 8 mg/kg. In one embodiment, the methodof the invention comprises administering a dose of the protein asdescribed in the present invention of about 0.5 mg/kg, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49 or of about 50 mg/kg.

In one embodiment, the method of the invention comprises administering adose of the protein as described in the present invention ranging fromabout 30 mg to about 3000 mg, preferably ranging from about 60 mg toabout 2000 mg, more preferably of about 100 to about 1000 mg, and evenmore preferably of about 500 mg. In one embodiment, the method of theinvention comprises administering a dose of the protein as described inthe present invention of about 30, 60, 62.5, 90, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500 or of about 3000mg. In another embodiment, the method of the invention comprisesadministering a dose of the protein as described in the presentinvention ranging from about 100 mg to about 2000 mg, from about 125 mgto about 2000 mg, preferably from about 250 mg to about 1000 mg or fromabout 500 mg to about 1000 mg.

In one embodiment, the protein as described in the present invention isinjected, preferably by intravenous infusion.

In one embodiment, the method of the invention comprises administeringthe protein during about 2 hours, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or during about 12 hours.

In one embodiment, the method of the invention comprises continuouslyadministering the protein during at least 2 hours, preferably during atleast 4 to 6 hours (e.g., during about 2 hours, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or during about12 hours).

In another embodiment, the method of the invention comprisesadministering a first bolus of the protein, followed by the continuousadministration of the remaining dose of the protein.

In one embodiment, said first bolus administration comprises about 10 to50%, preferably about 20% of the total dosage of the isolated humanizedprotein to be administered. In one embodiment, said first bolusadministration comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50% of the totaldosage of the isolated humanized protein to be administered.

In one embodiment, said first bolus is administered in about 5 to 30minutes, preferably in about 15 minutes.

In one embodiment, about 20% of the total dosage of the protein for usein the present administration are administered during the first 15minutes of the administration, followed by a continuous administrationof the remaining 80% during the next 5 hours 45 minutes.

In one embodiment, the method of the invention is for inhibiting GPVIreceptor function and downstream signalling, thereby treating a GPVIrelated condition.

In one embodiment, the method for inhibiting GPVI receptor function anddownstream signalling does not impact platelet count, expression of GPVIat the platelet surface nor bleeding time.

In one embodiment, the method for inhibiting GPVI receptor function anddownstream signalling is efficient and reversible.

In one embodiment, the method of the invention is for inhibiting thebinding of GPVI to its ligands (preferably, but not exclusively,collagen), thereby treating a GPVI related condition.

In one embodiment, the method for inhibiting the binding of GPVI to itsligands does not impact platelet count, expression of GPVI at theplatelet surface nor bleeding time.

In one embodiment, the method for inhibiting the binding of GPVI to itsligands is efficient and reversible.

In one embodiment, the method of the invention is for inhibiting and/orpreventing platelet adhesion to collagen, thereby treating a GPVIrelated condition.

In one embodiment, the method of the invention is for inhibiting and/orpreventing collagen-induced platelet aggregation, thereby treating aGPVI related condition

In one embodiment, the method of the invention is for inhibiting and/orpreventing platelet activation, in particular platelet aggregation, inresponse to collagen, thereby treating a GPVI related condition.

In one embodiment, the method of the invention is for inhibiting and/orpreventing thrombin production in response to collagen and/or to tissuefactor, thereby treating a GPVI related condition.

In one embodiment, the method of the invention is for inhibiting thebinding of GPVI to fibrin, thereby treating a GPVI related condition.

In one embodiment, the method of the invention is for inhibiting and/orpreventing platelet recruitment by fibrin via GPVI, thereby treating aGPVI related condition.

In one embodiment, the method of the invention is for inhibiting and/orpreventing GPVI-dependent thrombin production in response to fibrin,thereby treating a GPVI related condition.

In one embodiment, administering a protein as described hereinabove to asubject does not induce depletion of GPVI in vivo.

In one embodiment, administering a protein as described hereinabove to asubject does not induce a decrease in platelet count. Thus, in oneembodiment, administering a protein as described hereinabove to asubject does not induce thrombocytopenia.

In one embodiment, administering a protein as described hereinabove to asubject does not induce an increase in bleeding time.

In one embodiment, the method of the invention comprises administering atherapeutically effective amount of the protein to the subject.

The present invention further relates to a method for enhancing thepotency of a thrombolytic agent (preferably t-PA (including native t-PAand recombinant t-PA, as well as modified forms of t-PA that retain theenzymatic and/or fibrinolytic activity of native t-PA)) for treating aGPVI-related disease, wherein said method comprises administering acombination of said thrombolytic agent with a protein of the invention(preferably a therapeutically effective amount of the protein of theinvention) to the subject.

In one embodiment, the method of the invention allows decreasing thedose of thrombolytic agent (preferably t-PA) to be administered to thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are a combination of graphs showing the mean±SEM intensity(FIG. 1A) and velocity (FIG. 1B) of collagen-induced plateletaggregation measured 30 minutes and 2 hours after the end of a 15minutes' infusion of increasing doses of ACT017 (1, 2, 4, 8 mg/kg) tocynomolgus monkeys (n=4).

FIG. 2 is a graph showing the blood platelet count of cynomolgusmeasured before any treatment (TO) or 24 hours after the infusion ofvehicle or ACT017 at increasing doses (1, 2, 4, 8 mg/kg).

FIG. 3 is a combination of graphs showing the bleeding time measured in4 subjects, before treatment (Time=0) and 30 min and 2 hours after theinfusion of ACT017 (1, 2, 4, 8 mg/kg) or vehicle to cynomolgus monkeys.

FIGS. 4A-4B are a combination of graphs showing the mean±SD intensity(FIG. 4A) and velocity (FIG. 4B) of collagen-induced plateletaggregation measured at different time after the beginning of a one-hourinfusion of two doses of ACT017 (2 and 8 mg/kg) to cynomolgus monkeys(n=8).

FIG. 5 is a graph showing the level of GPVI expression measured by flowcytometry on the platelets of cynomolgus monkeys (n=8) at differenttimes after the beginning of a one-hour infusion of ACT017 at 2 mg/kg(MFI: Mean Fluorescence Intensity).

FIG. 6 is a graph showing the mean±SD intensity of collagen-inducedplatelet aggregation measured at different times (from 20 minutes to 24hours), after the end of a 25-minute bolus injection of ACT017 8 mg/kg(n=4), a 1-hour infusion of ACT017 8 mg/kg (n=8), or a 15-minute bolusof ACT017 2 mg/kg followed by a 5-hour-45-minute infusion of ACT017 6mg/kg to cynomolgus monkeys (n=4).

FIG. 7 is a graph showing the binding of wild-type GPVI-Fc (black graph)and GPVI double mutant (grey graph) on collagen.

FIG. 8 is a graph showing the binding of ACT017 on wild-type GPVI-Fc(black graph) or on double mutant (grey graph).

FIG. 9 is a graph showing the percentage of platelet aggregationmeasured with two healthy subjects of cohort 4 at different times (from15 min to 168 hours). Subjects of cohort 4 were administered with 500 mgof ACT017 or of placebo. The “screening time” represents the measurementof the percentage of platelet aggregation one week or 24 hours beforethe administration of ACT017 or its matching placebo. The “pre-dose”represents the baseline measurement of the administration of the ACT017or its matching placebo. In black is presented the graph for healthysubject 1 and in grey is presented the graph for healthy subject 2.

EXAMPLES

The present invention is further illustrated by the following examples.

Example 1: Biologic Data in Non-Human Primates

Materials and Methods

Animals

28 cynomolgus monkeys free of any previous treatment were used in thestudy.

Treatment

First, increasing doses of ACT017 (1, 2, 4, 8 mg/kg) or its vehicle wereintravenously administrated over 15 minutes (n=4-8). At time 30 minutesand 2 hours after administration, blood was collected.

In a second experiment, ACT017 was administered to animals, by bolusinjection or infusion. Three treatment groups were included in thestudy: ACT017 administrated at 8 mg/kg by a bolus injection of 25minutes (n=4), ACT017 administrated at 8 mg/kg by an infusion of 1 hour(n=8), ACT017 administrated at 2 mg/kg by a 15-min bolus injectionfollowed by a 6 mg/kg infusion of 5 hours and 45 minutes (n=4). Atdifferent times, from 20 minutes to 24 hours after administration, bloodwas collected.

Analysis

Platelet aggregation: Platelet rich plasma (PRP) was obtained frommonkeys after centrifugation (120×g; 15 min; 20° C.) and immediatelyused. The velocity and intensity of the aggregation were measured usingthe APACT® aggregometer and a collagen concentration of 2.5 mg. mL-1(Horm collagen, Nycomed, DE) to the PRP containing various concentrationof the Fab of the invention and continuously recorded. The intensity ofplatelet aggregation was measured as the percent increase in lighttransmission. The mean±SD are presented at the indicated time after thebeginning of the injection.

Platelet count was measured in EDTA anticoagulated blood. The plateletcount was determined in a Scil Vet abc automatic cell counter (ScilAnimal Care Company, Holtzheim, France) set to monkey parameters.

The bleeding time was measured on vigil monkeys at the surface of theforearm, according to standard clinical procedure (Ivy's procedure) andusing 0.5 cm Surgicutt™ bleeding time device.

GPVI expression: Blood samples were collected onto EDTA before injectionand 30 minutes post injection of increasing doses of ACT017 or anequivalent volume of vehicle and were incubated with commercial FITCcoupled anti-human GPVI monoclonal antibody (clone 1G5, Biocytex) thatcross react with cynomolgus GPVI, and fluorescence was measured on aBeckman Coulter Gallios Flow cytometer.

Results

The effect of ACT017 administration was characterized in non-humanprimates. Eight cynomolgus monkeys were enrolled in the study.

First, increasing doses of ACT017 (1, 2, 4, 8 mg/kg) were intravenouslyadministrated over 15 minutes. At time 30 minutes and 2 hours afteradministration, blood was collected. Platelet aggregation was reversiblyinhibited in a dose dependent manner (FIGS. 1A-1B). Increasing the dosefrom 1 to 2 and from 2 to 4 mg/kg increased the effect whereas 8 mg/kghad no additional effect as compared to 4 mg/kg. The platelet countmeasured 24 hours after the injection was not modified as compared tothe values obtained before injection (FIG. 2 ). No significant increasein the bleeding time was observed after treatment with vehicle or 2, 4or 8 mg/kg ACT017 (FIG. 3 ). Next, after a washing out period, thecynomolgus received ACT017 (2 or 8 mg/kg) administered as a one-hourperfusion. Platelet aggregation and GPVI expression were measured atdifferent time after the beginning of the treatment: (1, 1.5, 2, 4 and 7hours) (FIGS. 4 and 5 ). Collagen-induced platelet aggregation wasreversibly inhibited and the duration of the effect was prolonged incynomolgus treated with 8 mg/kg compared to 2 mg/kg (FIGS. 4A-4B). GPVIexpression on platelets remained stable at the different time after thebeginning of the treatment as compared to the pretreatment values (FIG.5 ).

Together, these results confirm in non-human primates thatadministration of ACT017 efficiently and reversibly inhibits GPVIfunction without impact on the platelet count, on expression of GPVI atthe platelet surface nor on bleeding time.

In addition, according to the preceding results, the inhibitory effectof ACT017 on platelet aggregation, after a 1-hour infusion appeared tobe efficient during a limited period of time. Indeed, ACT017 induced apronounced and stable inhibition on platelet aggregation only during 2hours after the beginning of the administration, i.e. for at most 1 hourafter the end of the 1-hour induction (see FIGS. 4A-4B). After 2 hours,even if the inhibition was prolonged at 8 mg/kg, ACT017 inhibitor effectregressed at all tested doses.

In a second experiment the effect of ACT017 at the dose of 8 mg/kg wasfurther characterized on platelet aggregation after differentadministration times, including 1-hour and 6-hour administrations.Platelet aggregation was reversibly inhibited in a time dependent manner(FIG. 6 ).

A 25-minute bolus injection at 8 mg/kg induced a full inhibition of thecollagen-induced platelet aggregation measured at 0.5 hour. At 2 hours,the inhibition of platelet aggregation regressed and the aggregationintensity returned to values of 24±34%. No effect of ACT017 can beobserved at 24h.

A 1-hour infusion at 8 mg/kg resulted in a prolonged inhibition ofcollagen-induced platelet aggregation. However, the inhibition was total(<5%) for all animals only until 2 hours. At 4 hours and 7 hours afterACT017 administration, inhibition regressed and the overall meanintensity of aggregation was respectively 16±29% and 25±32%. Therefore,as for the 25-min administration, the inhibitory effect of ACT017appears to be pronounced only during a short time after its 1-houradministration.

A 6-hour infusion at 8 mg/kg resulted in a profound inhibition ofcollagen-induced platelet aggregation lasting at least 9 hours. Indeed,during 9 hours after the beginning of the 6-hour infusion, plateletaggregation intensity was lower than 2%. Moreover, the possibility of alonger duration of the effect is not excluded since no analysis wasperformed between 9 hours and 24 hours. At 24 hours, the effect ofACT017 was close to be fully reversed for three out of the four animalsand the mean intensity of aggregation reached 47±30%.

In conclusion, for the same dose of 8 mg/kg, a 6-hour infusiondemonstrates a prolonged efficacy to inhibit collagen-induced plateletaggregation during at least 3 hours after the end of the administration.

Example 2: Study of ACTO17 Safety, Tolerability, Pharmacokinetic andPharmacodynamic in Healthy Volunteers

Materials and Methods

The present study is a randomized, double blind, placebo-controlledascending single dose study on the safety, tolerability,pharmacokinetics and pharmacodynamics of ACT017 in healthy volunteers.

Subjects

The subjects included in the study were healthy male or non-pregnant,non-breastfeeding female subject, aged between 30 and 60 year of age(inclusive) with a BMI≥18 kg/m2 and ≤30 kg/m2. A total of 48 subjectswere enrolled in 6 ascending dose level cohorts with each cohortconsisting of 8 subjects: 6 on active and 2 on placebo. Each cohort wasdivided into 2 sub-cohorts: one cohort dosed initially (1 active and 1placebo) and the other cohort (5 active and 1 placebo) 48 hoursthereafter.

On Day −1 of each dosing period, subjects were admitted to the researchcenter and stay there until at least 48 hours' post-dose. A follow upvisit was paid on Day 7. Subject were hospitalized from Day-1 until themorning of Day-3.

Treatment

Investigational drugs were 50 mL vial containing 500 mg of ACT017 andmatching placebo solution. Treatments were given during an infusion of 6hours.

Each dose was given as a 6-hour infusion, with a 15 minutes injection ofa first bolus corresponding to about ¼ of the final dosage.

Cohort 1: Treatment A: 62.5 mg ACT017 (n=6), Treatment B: matchingplacebo (n=2).

Cohort 2: Treatment A: 125 mg ACT017 (n=6), Treatment B: matchingplacebo (n=2).

Cohort 3: Treatment A: 250 mg ACT017 (n=6), Treatment B: matchingplacebo (n=2).

Cohort 4: Treatment A: 500 mg ACT017 (n=6), Treatment B: matchingplacebo (n=2).

Cohort 5: Treatment A: 1000 mg ACT017 (n=6), Treatment B: matchingplacebo (n=2).

Cohort 6: Treatment A: 2000 mg ACT017 (n=6), Treatment B: matchingplacebo (n=2).

Analysis

Pharmacokinetic blood sampling for ACT017 implied 15 samples persubject.

Urine collection for ACT017 implied approximately 5 collection intervalsper subject.

Safety and tolerability analysis included the following studies:

-   -   Adverse events: throughout the study;    -   Vital signs: frequently;    -   ECG (electrocardiogram): less frequent than vital signs;    -   Clinical laboratory including hematology: 2 times;    -   Coagulation parameters: 5 times;    -   Bleeding time: 4 times;    -   Platelet count: 7 times;    -   Platelet aggregation (collagen): 6 times;    -   Immunogenicity/ADA: 3 times.

Platelet aggregation: Platelet rich plasma (PRP) was obtained fromhealthy subjects having received the placebo or 500 mg of ACT017intravenously with 25% of the dose administered in the first 15 min an75% of the dose administered in the following 5 h 45 min, aftercentrifugation (120×g; 15 min; 20° C.) and immediately used. Theintensity of the aggregation were measured using the APACT® aggregometerand a collagen concentration of 2.5 mg. mL-1 (Horm collagen, variousconcentration of the Fab of the invention and continuously recorded. Theintensity of platelet aggregation was measured as the percent increasein light transmission.

Results

As shown by FIG. 9 , the percentage of aggregation of platelets insubject 2 decreases by about 70% only 15 min after the beginning of theadministrating in comparison with the baseline percentage and ismaintained at about 10% for the next 8 hours, then it is increased againto reach about 70% of aggregation after 24 hours, and then returns tobaseline 48 hours after administration. No effect is seen on plateletaggregation 168 hours after administration. The effect observed insubject 2 is thus reversible. In subject 1, the percentage of plateletaggregation seems unchanged during the 168 hours of the experience.

Example 3: Study of Epitopes Affinity of ACT017 on GPVI

The epitope of ACT017 on GPVI was previously identified by epitopemapping as being a conformational epitope comprising two regions onGPVI: amino acids 121-135 and amino acids 169-183 in SEQ ID NO: 13. Inorder to validate this epitope, a double mutant was constructed,comprising the following mutations: S125P, S126Q, G128R, Q133K, T136S,T171D, A172L and H174V and the affinity of this mutant for collagen andACT017 was measured.

Materials and Methods

Soluble GPVI-Fc was produced as follows: a gene encoding the ectodomainof human GPVI from the first methionine to asparagine 269 fused to thehuman IgG1 Fc domain via the tripeptide GGR was synthesized after codonoptimization. This gene was cloned into the pTT5 vector before beingtransfected into HEK 293-6E cells. Secreted GPVI-Fc was purified fromthe conditioned media of the cells by affinity chromatography usingMAbselect matrix (GE Healthcare, 17519901) followed by a polishingchromatography on Nuvia™ HR-S cation exchange resin (BioRad, 156051).

Effect of Double Mutant on Binding of GPVI-Fc on Collagen—

Microtitration plates were coated with Collagen in PBS (20 μg/mL, 100 μLper well) overnight at 4° C.

Nonspecific binding sites were saturated with 200 μL of 1% BSA in PBSfor 30 min. The plates were then incubated with increasingconcentrations of wild-type or double mutant GPVI-Fc preparations (100μL in PBS containing 0.1% BSA and 0.1% Tween 20) for 30 min. After 3washing rounds plates were incubated with a peroxidase-coupled secondaryhuman anti-Fc(ab) for 30 min. Finally, 100 μL of the substrate solutionwere added to each well for 4 min, and the colorimetric reaction stoppedby 25 μL NaOH 3 M.

Effect of Double Mutant on Binding of ACT017 on GPVI-Fc—

Microtitration plates were coated with GPVI-Fc in PBS (20 μg/mL, 100 μLper well) overnight at 4° C. Nonspecific binding sites were saturatedwith 200 μL of 1% BSA in PBS for 30 min. The plates were then incubatedwith increasing concentrations of ACT017 preparations (100 μL in PBScontaining 0.1% BSA and 0.1% Tween 20) for 30 min. After 3 washingrounds plates were incubated with a peroxidase-coupled secondary humananti-IgG for 30 min. Finally, 100 μL of the substrate solution wereadded to each well for 4 min, and the colorimetric reaction stopped by25 μL NaOH 3 M.

Analysis

Absorbance at 450 nm is measured with Flustar Optima.

Results

As shown by FIG. 7 , double mutant of GPVI-Fc is still capable to bindto collagen in a dose-dependent manner showing that the affinity of GPVIfor collagen is mainly conserved in the presence of these mutations.However, as shown by FIG. 8 , ACT017 is capable to bind wild-typeGPVI-Fc, but not the double mutant, even at high concentrations. Theseresults thus confirmed the position of the epitope of ACT017 on GPVIsequence.

The invention claimed is:
 1. A method for treating a cardiovasculardisease or event associated with inflammation and/or thrombosis in asubject in need thereof comprising administering an isolated humanizedprotein binding to human Glycoprotein VI (hGPVI), wherein said isolatedhumanized protein is administered for at least 2 hours, wherein saidisolated humanized protein is a monovalent antibody fragment, whereinthe variable region of the heavy chain of the antibody fragmentcomprises the following CDRs: VH-CDR1: (SEQ ID NO: 1) GYTFTSYNMH;VH-CDR2: (SEQ ID NO: 2) GIYPGNGDTSYNQKFQG; and VH-CDR3: (SEQ ID NO: 3)GTVVGDWYFDV;

and the variable region of the light chain of the antibody fragmentcomprises the following CDRs: VL-CDR1: (SEQ ID NO: 4) RSSQSLENSNGNTYLN;VL-CDR2: (SEQ ID NO: 5) RVSNRFS; and VL-CDR3: (SEQ ID NO: 6) LQLTHVPWT.


2. The method according to claim 1, wherein said isolated humanizedprotein is administered during at least 4 to 6 hours.
 3. The methodaccording to claim 1, wherein the isolated humanized protein isinjected.
 4. The method according to claim 1, wherein the isolatedhumanized protein is administered by intravenous infusion.
 5. The methodaccording to claim 1, wherein a dose of humanized protein ranging fromabout 0.5 mg/kg to about 50 mg/kg is administered to the patient.
 6. Themethod according to claim 1, wherein a dose of humanized protein rangingfrom about 1 mg/kg to about 32 mg/kg is administered to the patient. 7.The method according to claim 1, wherein a dose of humanized protein ofabout 8 mg/kg is administered to the patient.
 8. The method according toclaim 1, wherein a dose of humanized protein ranging from about 125 mgto about 2000 mg is administered to the patient.
 9. The method accordingto claim 1, wherein a first bolus is administered.
 10. The methodaccording to claim 9, wherein the first bolus comprises about 10 to 50%of the total dosage of the isolated humanized protein to beadministered.
 11. The method according to claim 9, wherein said firstbolus is administered in about 5 to 30 minutes.
 12. The method accordingto claim 1, wherein said protein binds to a conformational epitopecomprising: at least one amino acid residue from amino acid residues 114to 142 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60%of identity over amino acid residues 114 to 142 of hGPVI (SEQ ID NO:13); and at least one amino acid residue from amino acid residues 165 to187 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% ofidentity over amino acid residues 165 to 187 of hGPVI (SEQ ID NO: 13).13. The method according to claim 12, wherein said conformationalepitope comprises at least one amino acid residue from amino acidresidues 121 to 135 or from 121 to 136 of hGPVI (SEQ ID NO: 13) or froma sequence sharing at least 60% of identity over amino acid residues 121to 135 or from 121 to 136 of hGPVI (SEQ ID NO: 13); and at least oneamino acid residue from amino acid residues 169 to 183 of hGPVI (SEQ IDNO: 13) or from a sequence sharing at least 60% of identity over aminoacid residues 169 to 183 of hGPVI (SEQ ID NO: 13).
 14. The methodaccording to claim 1, wherein said protein has a KD for binding to hGPVIless than 15 nM, wherein said K_(D) is measured by surface plasmonresonance using 960 to 1071 RU of soluble human GPVI and using PBS pH7.4 as running buffer and wherein said isolated humanized protein doesnot induce a GPVI depletion phenotype in vivo.
 15. The method accordingto claim 1, wherein said protein is a monovalent antibody fragmentselected from the group consisting of a single chain antibody, a Fv, aFab; and a unibody.
 16. The method according to claim 1, wherein theamino acid sequence encoding the heavy chain variable region of theantibody fragment is SEQ ID NO: 7 and the amino acid sequence encodingthe light variable region of the antibody fragment is SEQ ID NO: 8, orany sequence having an amino acid sequence that shares at least 60% ofidentity with said SEQ ID NO: 7 or
 8. 17. The method according to claim15, wherein the amino acid sequence encoding the heavy chain variableregion of the antibody is SEQ ID NO: 7 and the amino acid sequenceencoding the light variable region of the antibody is SEQ ID NO: 9, orany sequence having an amino acid sequence that shares at least 60% ofidentity with said SEQ ID NO: 7 or
 9. 18. The method according to claim1, wherein said cardiovascular disease or event associated withinflammation and/or thrombosis is selected from arterial and venousthrombosis, restenosis, acute coronary syndrome, cerebrovascularaccidents due to atherosclerosis, critical limb ischemia, cerebralvascular diseases, ischemic stroke, venous thromboembolism diseases,thrombotic microangiopathies and vascular purpura.
 19. The methodaccording to claim 1, wherein said cardiovascular disease or eventassociated with inflammation and/or thrombosis is selected from coronaryartery and cerebral artery diseases.
 20. The method according to claim1, wherein said cardiovascular disease or event associated withinflammation and/or thrombosis is selected from atherothrombosis,ischemic events, acute coronary artery syndrome, myocardial infarction,stroke, percutaneous coronary intervention, stenting thrombosis,ischemic restenosis, acute ischemia, chronic ischemia, diseases of theaorta and its branches, peripheral artery disease, venous thrombosis,acute phlebitis, pulmonary embolism, cancer-associated thrombosis,inflammatory thrombosis and thrombosis associated to infection.